Proteinúria é a principal manifestação da nefrite lúpica (NL) e reflete lesão no podócito. Análise dos biomarcadores do podócito foi realizada com o objetivo de identificar se o fenótipo podocitário é distinto na NL membranosa pura e proliferativa. Expressão de sinaptopodina, proteína 1 do tumor de Wilms (Wilms tumor protein 1 - WT1), proteína epitelial glomerular 1 (glomerular epitelial protein 1 - GLEPP1) e nefrina foi avaliada em 52 biópsias de NL por imunohistoquímica. Expressão preservada de sinaptopodina foi observada em apenas 10 (19,2%) de todas as biópsias enquanto que 42 (80,8%) apresentavam expressão reduzida. Ambos os grupos tinham proteinúria semelhante no momento da biópsia (p = 0,22), porém, no seguimento médio de quatro anos houve uma tendência para menores níveis médios de proteinúria nos pacientes com marcação preservada de sinaptopodina (0,26 ± 0,23 vs 0,84 ± 0,90 g/24 h, p = 0,05) do que naqueles com expressão reduzida. Trinta e nove (75%) biópsias foram classificadas como proliferativa e treze (25%) como membranosa pura. Comparação dos biomarcadores do podócito demonstrou predomíno de marcação preservada de sinaptopodina (69,2%), WT1 (69,2%), GLEPP1 (53,9%) e nefrina (60%) no grupo membranosa pura enquanto apenas < 10% das proliferativas apresentaram expressão preservada. Nossos dados sugerem que nas classes proliferativas parece haver lesão estrutural do podócito, enquanto que na membranosa pura o padrão predominantemente preservado sugere uma lesão funcional do podócito que pode ser responsável pelo melhor prognóstico a longo prazo do desfecho da proteinúria / Proteinuria is a major feature of lupus nephritis (LN) and reflects podocyte injury. Analysis of podocyte biomarkers was performed attempting to identify if podocyte phenotype is distinct in pure membranous and proliferative LN. Expression of synaptopodin, Wilms tumor protein 1 (WT1), glomerular epithelial protein 1 (GLEPP1) and nephrin was evaluated in 52 LN biopsies by immunohistochemistry. Preserved synaptopodin expression was observed in only 10 (19,2%) of all biopsies while 42 (80,8%) had a reduced expression. Both groups had comparable proteinuria at the time of biopsy (p=0,22), however, in the mean follow-up of four years there was a tendency to lower mean levels of proteinuria in patients with preserved synaptopodin staining (0,26 ± 0,23 vs. 0,84 ± 0,90 g/24 h, p=0,05) than those with diminished expression. Thirty-nine (75%) biopsies were classified as proliferative and thirteen (25%) as pure membranous. Comparison of podocyte biomarkers demonstrated a predominance of preserved staining of synaptopodin (69,2%), WT1 (69,2%), GLEPP1 (53,9%) and nephrin (60%) in the pure membranous group whereas only < 10% of the proliferative showed preserved expression. Our data suggest that in proliferative forms there seems to occur structural podocyte damage, whereas in the pure membranous the predominant preserved pattern suggests a dysfunctional podocyte lesion that may account for the better long-term prognosis of proteinuria outcome

The purpose of this study was 1) to examine the significance of a patient’s active or passive role in terms of his/her health management; 2) to determine if a relationship exists between one’s active and passive scores and his/her self-directed learning readiness, and 3) to identify if his/her view of one’s self as a patient (when diagnosed with a chronic disease) impacted his/her own personal health management. Utilizing the quantitative analysis of The Self-Directed Learning Readiness Scale and the Vanderbilt Pain Management Inventory, 81 individuals’ descriptive statistics were analyzed. Self-directed learning was found to positively influence an individual’s ability to be an active patient. The moderated demographic characteristics of age, ethnicity, education level, and gender did not have a direct relationship between selfdirected learning readiness and active/passive coping groups. Further, it was established that the majority of the participants within the study, 83.75%, considered themselves an active patient managing their lupus diagnosis. However, 16.25% of the participants did not believe that they were actively managing their illness. Self-directed learning characteristics were examined through the responses to an open-ended question. The two most prevalent themes pertained to active coping and control. Characteristics of self-directed learning readiness appeared predominant amongst the responses, particularly goal-orientation and accepting responsibility for learning. Minimal themes regarding planning and enjoying learning were provided within the data. These characteristics were identified throughout the study in hopes of further research and program implementations that will help to develop leadership abilities and activity levels of self-health management in chronically ill patients. This will enable lupus patients to have a more positive outcome, it will help them successfully manage their own health, and it will improve their overall quality of life. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

This study evaluated two methods for the isolation and purification of anti-DNA antibodies. A two-step affinity purification with streptavidin (SA) biotinylated oligodeoxythymidine (dT) M-280 and protein G Dynabeadsª was compared to a two step method using Melon(TM) Gel and cellulose DNA. Although Melon gel allowed for faster antibody purification and a higher recovery rate it gave a product of less purity than the magnetic bead method. Further characterization of the antibodies was done by PhastGel(TM) non-reducing SDS-PAGE and isoelectric focusing in order to analyze purity and confirm the polyclonal nature of anti-DNA antibodies. Agilent 2100, with a higher resolution then SDS-PAGE, revealed possible subclasses of different MW not detected by SDS-PAGE. ELISA showed that all four IgG antibody subclasses were present, while Western blot confirmed the presence of human IgGs. Ultraviolet spectroscopy, Agilent, and fluorescence based assays were used to demonstrate DNA hydrolytic activity of purified anti-DNA antibody. / by Anna M. Kats. / Thesis (M.S.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, FL : 2008 Mode of access: World Wide Web.

O lúpus eritematoso sistêmico (LES) é uma doença autoimune caracterizada por hiperatividade imunológica crônica pela ação de autoanticorpos que afetam diversos órgãos. Embora o uso crônico de imunossupressores predisponha o paciente a infecções, poucas pesquisas avaliaram uma possível associação entre doença periodontal e LES. Os objetivos deste trabalho foram investigar, por meio de estudo caso-controle, a prevalência e a gravidade da doença periodontal em pacientes com LES, e identificar e quantificar as principais bactérias periodontais presentes no biofilme subgengival. Foram incluídas 60 mulheres em atendimento no Setor de Reumatologia do Hospital Universitário de Brasília, de 20 a 65 anos de idade, sendo subdivididas em LES-A (ativo; n= 31) e LES-I (inativo; n=29). O grupo controle foi composto por 31 mulheres com os mesmos critérios de inclusão, porém sem doenças sistêmicas. As pacientes foram avaliadas quanto às medidas de profundidade de sondagem (P.S), perda de inserção clínica (PIC), índice de sangramento do sulco (ISS) e índice de placa (IPl) no exame inicial. Foram coletados biofilmes subgengivais dos quatro sítios mais profundos para identificação e quantificação de periodontopatógenos por meio de hibridação DNA-DNA checkerboard. Não houve diferenças estatisticamente entre os grupos relativamente aos parâmetros clínicos periodontais, exceto para o ISS, que foi menor no LESA (11,19% ± 14,62%) comparativamente ao grupo controle (17,30% ± 14,88%), porém sem diferenças quando comparado com o grupo LES-I (11,34% ± 11,59%). Houve baixa prevalência de bolsas periodontais, de PIC 4 mm e de espécies de Actinomyces em todos os grupos. Verificou-se aumento na contagem de bactérias do complexo vermelho no grupo LES-I (4,07 x 105; 95% CI: 0,16-0,79) em relação ao grupo controle (2,50 x 105; 95% CI: 1,23-3,77), com diferenças estatisticamente significante apenas referente ao grupo LES-A (p< 0,05; Kruskal Wallis pós-teste Dunn; 0,45 x 105; 95% CI: 0,16-0,79). Os resultados desse estudo demonstraram que os parâmetros periodontais são semelhantes entre pacientes com LES e grupo controle. O grupo de LES-I apresentou maior tempo dessa doença; aumento da contagem de microorganismos (especialmente dos complexos vermelho e verde em amostras de biofilme subgengival) e pior condição periodontal. / Systemic lupus erythematous (SLE) is an autoimmune disorder characterized by chronic immunological hyperactivity resultant from the action of autoantibodies, affecting many organs. Although the use of immune suppressors may predispose infections, few studies have investigated the prevalence and severity of periodontal disease in SLE patients. The aim of this study is to investigate the prevalence and severity of periodontal disease in SLE patients and the sub gingival levels of different pathogens. A total of 60 women attending of Brasília University Hospital, aged 18-65 years, were invited to participate in the study. SLE patients were allocated in two subgroups according with disease activity: SLE-A (active disease; n= 31) and SLE-I (inactive disease; n= 29). A number of 31 systemically healthy women at the same age range composed control group. Patients were clinically examined according to probing depth (PD), clinical attachment level (CAL), sulcular bleeding index (SBI) and plaque index (PLI) at baseline examination. Sub gingival biofilm samples were collected from the deepest four sites before periodontal treatment in order to identify and quantify the level of periodontopathogens by checkerboard DNA-DNA hybridization. No significant differences were found between groups in PD, CAL and PLI. Significant differences were observed in GBI between SLE-A (11,19% ± 14,62%) compared to controls (17,30% ± 14.88%), although with no differences when compared to SLE-I (11,34% ± 11,59%). There was a low prevalence of PD and attachment loss 4 mm at all groups. A low prevalence of Actinomyces was observed at all groups, with an increase in red complex species at LES-I (4,07 x 105; 95% CI: 0,16-0,79) compared to control (2,50 x 105; 95% CI: 1,23-3,77), although with significant differences (p< 0,05; Kruskal Wallis post hoc Dunn) only when compared to SLE-A (0,45 x 105; 95% CI: 0,16-0,79). These findings show no differences in the periodontal conditions of SLE compared to systemically healthy patients, except for a decrease in gingival bleeding index, especially at SLE-A. Reductions in microorganisms count were observed at SLE-A, while an increase in bacterial count, especially at red and green complex, were observed at subgingival biofilm of SLE-I patients.

O lúpus eritematoso sistêmico (LES) é uma doença autoimune e protótipo das doenças por imunocomplexos (IC). A deposição de IC em tecidos ou órgãos leva a um processo inflamatório crônico, que resulta em lesão tecidual. A fisiopatologia deste processo envolve principalmente o sistema do complemento (SC), receptores de IgG (Fc?R), neutrófilos e moléculas de adesão. Os produtos de ativação SC atraem os neutrófilos para o foco inflamatório e sua interação com os IC depositados resulta na liberação de espécies reativas de oxigênio (ERO) e das enzimas dos neutrófilos sobre os tecidos. As ERO podem levar a danos nas estruturas celulares devido ao estresse oxidativo, resultando na exposição de autoantígenos e sustentação da autoimunidade. Várias anormalidades envolvendo neutrófilos, função e expressão dos Fc?R e CR foram descritas no LES. O objetivo deste estudo foi avaliar se essas alterações podem ter implicações para o dano tecidual através do depósito de IC. Devido à importância das interações neutrófilo-endotélio-SC para a inflamação e lesão tecidual por IC no LES, este estudo propôs avaliar o efeito da ativação de neutrófilos e produtos do SC sobre as células endoteliais in vitro. Os resultados mostram que a exposição das células endoteliais aos neutrófilos/LES ativo, sem estímulo, resulta em maior peroxidação lipídica comparada com o controle espontâneo, o que não foi observado com neutrófilos/saudáveis. Contudo, na presença de IC/SHN, a peroxidação lipídica foi maior quando as células endoteliais foram expostas aos neutrófilos/LES inativo comparada ao controle espontâneo. O efeito da ativação dos neutrófilos, em todos os grupos, sobre a peroxidação lipídica das células endoteliais foi dependente da opsonização dos IC pelo complemento (IC/SHN), uma vez que não foi observado quando as proteínas do complemento foram inativadas (IC/SHI). Não houve diferença na produção de ERO entre os neutrófilos dos grupos estudados. Observou-se menor expressão dos Fc?RIIa (CD32) e CR1 em neutrófilos/LES ativo, quando comparados com o grupo controle. Houve maior liberação de catalase pelos neutrófilos/LES, quando estes foram estimulados via Fc?R, e maior produção de glutationa por neutrófilos/LES inativo quando estas células foram estimuladas via Fc?R e CR. A expressão de ICAM-1 não foi diferente entre os grupos de neutrófilos, entretanto foi menor na ausência de complemento. Não houve diferença na medida da ativação da via clássica do complemento avaliada pelo fragmento C4d. Estes resultados mostram que as células endoteliais são mais suscetíveis à peroxidação lipídica na presença de neutrófilos/LES. Contudo, quando o complemento do soro é inativado, esta suscetibilidade desaparece, bem como há menor liberação de ICAM-1 por todos os grupos de neutrófilos e maior liberação de catalase por neutrófilos/LES. A interação dos neutrófilos/LES com células endoteliais pode ser deletéria para este último e depender da atividade das proteínas do sistema complemento. O modelo desenvolvido neste estudo pode contribuir para a compreensão do envolvimento dos neutrófilos e do SC nas lesões teciduais no LES. / Systemic lupus erythematosus (SLE) is an autoimmune disease and prototype of immune complex diseases (IC). The deposition of IC in tissues or organs leads to a chronic inflammatory process, which results in tissue injury. The pathophysiology of this process mainly involves the complement system (SC), IgG (Fc?R) receptors, neutrophils and adhesion molecules. SC activation products attract neutrophils to the inflammatory focus and their interaction with deposited IC results in the release of reactive oxygen species (ROS) and neutrophil enzymes into tissues. ROS can lead to damage to cell structures due to oxidative stress, resulting in the exposure of autoantigens and the support of autoimmunity. Several abnormalities involving neutrophils, function and expression of Fc?R and CR have been described in SLE. The aim of this study was to assess whether these changes may have implications for tissue damage through the deposition of IC. Due to the importance of neutrophil-endothelial-SC interactions for inflammation and tissue damage by IC in SLE, this study proposed to evaluate the effect of the activation of neutrophils and SC products on endothelial cells in vitro. The results show that exposure of endothelial cells to active neutrophils / SLE without stimulus results in higher lipid peroxidation compared to spontaneous control, which was not observed with neutrophils / healthy. However, in the presence of IC / complement from normal human serum (NHS), lipid peroxidation was greater when endothelial cells were exposed to inactive neutrophils / SLE compared to spontaneous control. The effect of neutrophil activation in all groups on endothelial cell lipid peroxidation was dependent on IC with complement (IC / NHS), as it was not observed when complement proteins were inactivated (IC / INHS). There was no difference in ROS production among the neutrophils of the studied groups. Lower expression of Fc?RIIa (CD32) and CR1 in active neutrophils / SLE, when compared with the control group, was observed. There was greater release of catalase by neutrophils / SLE, when they were stimulated via Fc?R, and increased production of glutathione by neutrophils / inactive SLE when these cells were stimulated via Fc?R and CR. There was no difference in expression of ICAM-1 between the neutrophil groups, however it was lower in the absence of complement. There was no difference in the extent of the activation of the classical complement pathway evaluated by the C4d fragment. These results show that endothelial cells are more susceptible to lipid peroxidation in the presence of neutrophils / SLE. However, when serum complement is inactivated, this susceptibility disappears, as well as there is a lower release of ICAM-1 by all neutrophil groups and greater release of catalase by neutrophils / SLE. The interaction of neutrophils / SLE with endothelial cells may be deleterious to the latter and depends on the activity of the complement system proteins. The model developed in this study may contribute to the understanding of neutrophil and SC involvement in tissue lesions in SLE.

From this series of studies, we conclude that microalbuminuria may represent a novel risk factor in SLE, and lupus patients are more susceptible to endothelial dysfunction caused by hyperhomocysteinemia. The use of antimalarial agents is beneficial for lupus patients with active disease on corticosteroid, and antioxidant vitamins are useful in lowering the oxidative stress markers but do not affect the endothelial function. The results highlight the importance of targeting the known modifiable risk factors in order to prevent premature atherosclerosis in SLE patients. / My first step was to elucidate the prevalence and metabolic abnormalities in SLE patients with microalbuminuria. Twenty percent of patients were found to have microalbuminuria, which was associated with higher mean arterial pressure, total plasma antioxidant and homocysteine levels. / Next, we recruited 12 SLE patients and 15 controls and gave them oral methionine loading to achieve acute hyperhomocysteinemia. After oral methionine loading, von Willebrand factor (vWF) levels increased significantly in both groups. The increase in vWF was apparently more pronounced in SLE (20%) compared to controls (8%). Fibrinogen binding to platelets increased significantly only in SLE patients. / Systemic Lupus Erythematosus (SLE) is a chronic inflammatory disease of unknown cause which can affect any organs. Studies have reported an increased prevalence of cardiovascular disease (CVD) in these patients. We performed a series of studies to elucidate the interaction between microalbuminuria, dyslipidaemia, hyperhomocysteinemia, oxidative stress and immune dysregulation from the underlying disease in order to understand the accelerated atherosclerotic process in SLE. / We then evaluated the effects of long-term antioxidant vitamins. The plasma malondialdehyde level was significantly decreased after treatment in the vitamin group. Other oxidative stress markers and antioxidant levels and endothelial function remained unchanged in both groups. / We then proceeded to study the relative effect of antimalarial agents on fasting lipid fractions in patients with active SLE. Total cholesterol, very low-density lipoprotein cholesterol, and low density lipoprotein cholesterol levels were significantly lower in patients taking antimalarial agents, particularly for those patients taking concomitant prednisone. In the last study, we demonstrated that hydroxychloroquine had no significant effect on the serum lipid profile in these lupus patients with mild or inactive disease. / Tam Lai Shan. / Adviser: Edmund K. Li. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1550. / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 178-214). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

[pt] Focalizando o Lúpus Eritematoso Sistêmico como uma doença psicossomática, localizada no limiar entre o psíquico e o físico, esta dissertação pretende contribuir para o estudo do trauma, da dor psíquica e física, utilizando-se das teorias de Winnicott e Freud para este fim. Considerando que a dor física faz parte do quadro clínico do Lúpus Eritematoso Sistêmico, este trabalho levanta a questão de que o quadro álgico torna-se muitas vezes insuportável, por se somar à dor física, o eu que dói, decorrente de traumas vividos. O corpo adoecido e dolorido reclama a ausência de um meio ambiente bom o bastante levando a quebra na vivência de continuidade do ser, (Winnicott), assim como a ausência de uma barreira de estímulos (Freud) forte, que permita o desenvolvimento adequado do eu. Para ilustrar as idéias apresentadas no trabalho, foram retirados fragmentos dos relatos, realizados no ambulatório do Hospital Universitário Pedro Ernesto. / [en] Focusing Systemic Erythematosus as a psychosomatic disease, located in the difficult thesrhold between the psychic and the physical, this dissertation intends to contribute for the study of the trauma, of psychic and physical pain, using the theories of Winnicott and Freud for this purpose. Considering that physical pain is part of the clinical picture of Systemic Lupus Erythematosus, this work raises the subject that of pain becomes many times unbearable, considering that to physical pain, pain of the self is odded, due to past, experienced traumas. The sick and aching body claims the break in the experience of the continuity of being, lock of a good enough environment (Winnicott), as well as lock of a strong stimuli barrier (Freud), allowing for on appropriate development of the self. To illustrate the ideas presented in this work, fragments of patients reports from the clinic of the University Hospital, Pedro Ernesto, were used for this purpose.

研究背景 / 系統性紅斑狼瘡（SLE）是一種較常見的累及多系統多器官的自身免疫性疾病，由於細胞和體液免疫功能障礙，產生多種自身抗體，確切病因不明。研究一般認為是在遺傳、環境等諸多因素的共同作用下導致了機體固有免疫及獲得性免疫系統功能紊亂，從而發病。作為機體抵抗病原體入侵的第一道防線，固有免疫系統通過模式識別受體（PRRs），不僅可以識別結合外源性的病原體相關模式分子（PAMPs），也可識別機體自身細胞所釋放的內源性危險信號又稱破壞相關模式分子（DAMPs），從而啟動信號傳導途徑，激活天然免疫細胞，從而導致一系列免疫應答的發生。本文將初步探討三類PRRs在SLE發病機制及病毒感染中的作用：（i）胞質中識別細胞壁肽聚糖的NOD樣受體（NLR），（ii）識別危險信號分子（DAMP）的膜結合型晚期糖基化終末產物受體（RAGE）及（iii）識別核酸的胞內Toll樣受體（TLR）。 / NLR是一種新發現的PAMP識別受體，在配體識別及信號傳導方面有別於膜型PRRs，在固有免疫應答中發揮重要作用。目前報道中NLR至少有23個成員，其中最有代表性的是NOD1和NOD2，通過特異性識別細菌胞壁肽聚糖產物從而參與固有免疫應答並誘導炎症反應和細胞凋亡。前期研究多集中於NODs與SLE基因易感性的探討，而SLE患者體內免疫細胞是否功能性表達NOD2及其下遊 的效應如何，仍有待進一步探討。 / RAGE是一種多配體受體，廣泛分布於上皮細胞、血管及炎症細胞表面，低表達於正常組織細胞，但與其配體結合後可啟動激活細胞內部各種信號傳導機制從而產生相應的生物學效應。HMGB1作為RAGE的重要配體在細胞和組織中分佈十分廣泛，近年研究證實，胞外高表達的HMGB1為一種重要的內源性危險信號分子，通過RAGE受體通路，可促進趨化作用，並通過激活NF-κB途徑誘導炎症反應。越來越多的證據表明HMGB1在自身免疫性疾病中起積極作用， RAGE-HMGB1軸在SLE的炎症反應及組織損傷中的重要作用值得研究。 / RAGE基因可因RNA的選擇性剪接而分為：全長RAGE（flRAGE）、截去N端的RAGE及截去C端的可溶性RAGE（sRAGE）。sRAGE有兩種來源，其中由細胞分泌而來的又稱為內分泌性RAGE（esRAGE）。sRAGE通過與flRAGE競爭性與結合配體從可抑制RAGE誘導的細胞信號傳導途徑，故又稱為作為“誘餌受體，作為潛在的治療靶點，對疾病的進展有保護作用。因此，評估sRAGE/esRAGE與flRAGE及配體HMGB1在SLE患者體內的動態平衡具有顯著臨床意義。 / 人類乳頭狀病毒（HPV）感染是子宮頸癌的致病因素，高危型HPV感染的持續存在是子宮頸癌的重要風險因素之一。早期病例對照研究已提示伴高炎症狀態及長期多重高危型HPV感染的SLE患者其子宮頸巴氏塗片異常和宮頸癌的發病率顯著高於對照人群，但在前瞻性隊列研究中其致高危致病因素及預測因子並未得到證實。TLR家族在早期固有免疫中對入侵病原微生物的識別發揮重要作用，胞內TLR3、TLR7、TLR8、TLR9通過識別病毒核酸成分通過介導下遊信號轉導誘導免疫反應。是否固有免疫系統異常參與SLE患者體內HPV持續及高危感染，仍有待進一步探討。 / 研究目的 / 1.研究NOD2受體通路及RAGE-HMGB1軸在SLE發病機制中的作用； / 2.闡明sRAGE/esRAGE作為“保護因子與flRAGE及其配體HMGB1在SLE患者體內的動態平衡及評估與疾病活動相關性； / 3.探討TLR受體通路在宿主SLE抗 HPV感染中的作用。 / 研究方法 / 本文通過三項臨床病例對照研究分別探討NLR、RAGE、TLR在SLE發病機制及病毒感染中的作用。 / 研究結果 / 1.SLE外周高表達於單核細胞內的NOD2可通過特異性識別配體誘導外周血單個核細胞的異常活化及促炎細胞因子的產生；而免疫抑制治療可下調CD8+ T細胞及抗原體提呈細胞內NOD2表達及抗炎細胞因子的產生； / 2.FlRAG高表達於SLE患者外周血單核細胞；血漿sRAGE作為獨立風險因素，與SLE疾病活動指數呈負相關；HMGB1單獨或與TLR9配體協同作用可刺激單核細胞分泌促炎細胞因子並激活信號轉導通路； / 3.TLR拮抗劑（羥氯喹）及強的松治療作為獨立風險因素可下調SLE患者子宮頸上皮細胞中TLR7和TLR9的表達；腫瘤相關的高危型HPV細胞株內核酸識別受體TLR及幹擾素刺激基因（ISGs）表達明顯下調伴功能異常。 / 研究結論 / 一方面，異常活化的PRRs通過識別結合外源及內源性病原體相關分子從而啟動固有免疫應答，激活一系列信號轉導通路，參與SLE的自身免疫反應： / 1.胞內受體NOD2可能通過特異性識別細菌胞壁酰二肽及誘導炎症反應，參與固有免疫應答反應抵禦外源性病原體侵襲，為感染因素導致SLE發病的假說尋找進一步理論依據； / 2.膜表面受體RAGE與配體HMGB1結合可激活細胞內多信號轉導機制，參與固有免疫應答反應抵禦內源性病原體侵襲，為胞內危險信號分子释放導致SLE無菌性炎症的假說提供初步理論依據； / 3.可溶性“誘餌受體RAGE作為潛在治療靶點可抑制SLE體內高炎症反應。 / 另一方面，多重因素交叉作用可引起SLE患者體內PRR轉錄及表達下調，從而抑制固有免疫應答，導致病毒逃避宿主免疫系統的監視及清除而長期潛伏： / 1.TLR拮抗劑（羥氯喹）和強的松治療可能引起SLE患者體內TLR7和TLR9表達下調，從而抑制固有免疫系統對外源侵入性病原體HPV的識別； / 2.腫瘤相關的高危型HPV細胞株亦可通過抑制TLR7和TLR9轉錄、下調受體表達及功能致 HPV逃避宿主免疫防禦而長期潛伏。 / Introduction / The pathogenesis of systemic lupus erythematosus (SLE) is a complicated process caused by genetic and environmental factors resulting in abnormalities of both the innate and the adaptive immune system. Sensing the presence of a pathogen is the first step for the immune system to mount an effective response to eliminate invading microorganisms and establish protective immunity. The innate immune system constitutes an important defense system to respond rapidly to both endogenous and exogenous molecules, in which the pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs) can interact with the pattern recognition receptors (PRRs) and then activate the antigen presenting cells (APCs), T, B cells. / Effective sensing of endogenous and exogenous molecules promotes autoreactivity via immune activation and antigen presentation. In lupus, these molecules may have a special role in the pathogenesis since they can serve as targets of autoreactivity as well as inducers. In this series of experiments, we focused on the roles of various PRRs including nucleic acid sensing toll-like receptors (TLRs), bacterial peptidoglycans sensing NOD-like receptors (NLRs) and dangerous signals sensing receptor for advanced glycation end products (RAGE), which are involved in the recognition of PAMPs and DAMPs sharing between microbes and the host in the pathogenesis of SLE. / In contrast to the well elucidated membrane-bound TLRs, cytoplasmic NLRs are a new family of PRRs for the recognition of extracellular PAMPs. NLRs can participate in the signaling events triggered by host recognition of specific motifs of bacterial peptidoglycans and, upon activation, induce the production of proinflammatory mediators. Apart from the putative link between genetic mutations of NOD2 and SLE, little is known regarding the expression and function of NOD2 in SLE. / RAGE is a transmembrane cell-surface receptor on a variety of immune effector cells, which is expressed at low levels in normal tissues and vasculature, but is upregulated wherever the accumulation of its proinflammatory ligands, especially the key ligand, high mobility group box protein 1 (HMGB1). Both endogenous secretory RAGE (esRAGE) as well as soluble RAGE (sRAGE) can be detected in blood serum and are able to bind the circulating ligands, neutralizing their actions. In those conditions characterized by high concentrations of the circulating ligands, the decoy receptors are reduced drastically, revealing the system function. Therefore, the relationship between the upregulation of full-length (fl) RAGE/RAGE ligands and the levels of “protective“ esRAGE/sRAGE in SLE is of obvious clinical interest. / Apart from inducing and perpatating autoreactivity, abnormal innate response may also be responsible for the increased risk of infection in patients with lupus. The prevalence of abnormal Papanicolaou (Pap) smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk and multiple human papillomavirous (HPV) infections. However, none of the clinical, lifestyle, gynecological and treatment parameters was predictive of persistent HPV infection. Innate immune recognition of viral infection triggers antiviral immune responses. Whether the abnormal host innate immune response in lupus patients may play a role in enhancing HPV persistence remained unknown. / Hypothesis / 1.Aberrant activation of NLR and RAGE pathways by endogenous or exogenous ligands lead to the initation and/or perpetuation of autoimmune responses in SLE; / 2.HPV infection suppresses the host immune response by deregulating the TLRs transcript, leading to increased viral persistence in SLE. / Aims / 1.To evaluate the role of NOD2 pathway in the pathogenesis of SLE; / 2.To elucidate the relationship and regulatory mechanisms among members of the RAGE axis in the pathogenesis of SLE; / 3.To investigate the role of TLR in the defense against HPV infection in SLE. / Methods / The present thesis comprised of three cross-sectional studies in Chinese patients with SLE and controls in Hong Kong. Clinical assessments and review of medical records were performed to obtain information regarding disease status. / Results / 1.Over-expression of NOD2 in monocytes was observed in immunosuppressant naive SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8⁺ T, monocytes and dendritic cells (DCs). Ex vivo basal productions of cytokines [Interleukin (IL)-6, IL-8 and IL-10] were significantly increased in immunosuppressant naive patients and patients with active disease despite immunosuppressants compared with healthy controls. Upon muramyl dipeptide (MDP) stimulaiton, relative induction (%) of cytokines (IL-1β) from peripheral blood mononuclear cells (PBMCs) was significantly increased in immunosuppressant naive patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with healthy controls. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naive patients and healthy controls. / 2.Plasma sRAGE level was negatively correlated with SLE disease activities. The reduction in sRAGE levels in SLE patients with flare indicates that sRAGE may play a regulatory role on disease activity. HMGB1 alone could only mildly induce IL-6 production, which resulted in a transient phosporylation of intracellular p38 mitogen activated protein kinase (MAPK), c-Jun NH2- terminal protein kinase (JNK) and nuclear factor (NF)-κB. On the other hand, CpG-oligodeoxynucleotides (ODN) (TLR9 ligand) together with HMGB1 not only had a additive effect on IL-6 and IL-12p70 secretions compared with each agent alone, but also activated the phosphorylation of p38 MAPK and NF-κB. / 3.TLR inhibitor (hydroxychloroquine) and prednisolone may down-regulate protein levels of TLRs 7 and 9 in cervical epithelial cells of lupus patients. In the cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral interferon-stimulated genes (ISG) 15 and myxovirus resistance (Mx) 1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands [R837, single stranded (ss) RNA and CpG-ODN] was greatly impaired in CaSki and HeLa than C33A cells. / Conclusions / Aberrant activation of PRR pathways by endogenous or exogenous molecules triggers the initation and/or perpetuation of autoimmune responses as follows: / 1.NOD2 may participate in the pathogenesis of lupus via the recognition of MDP and induction of proinflammatory effects, implicating the innate immune response for endogenous pathogens in the immunopathological mechanisms in SLE; / 2.Over-expression of RAGE may amplify the pro-inflammatory effects of DAMP such as HMGB1, while soluble RAGE may serve as a decoy receptor to suppress inflammation in patients with lupus nephritis. / 3.Upregulated HMGB1 may act alone or in combine with TLR9 ligand through the phosphorylation of p38 MAPK and NF-κB to promote inflammation in lupus. / On the other hand, the immune evasion strategy via avoidance of stimulation and downregulation of PRRs may promote establishment of persistent infection as listed below: / 1.TLR inhibitor (hydroxychloroquine) and prednisolone may down-regulate protein levels of TLRs 7 and 9 in lupus patients, thereby decreasing the innate immune response against HPV infection. / 2.Upon infection, HPV further down-regulate TLRs 7 and 9 levels for viral persistence. / 3.Reduction of TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs on a biologically relevant antiviral response. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yu, Shuilian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 114-141). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.v / ACKNOWLEDGEMENTS --- p.viii / LIST OF PUBLICATIONS --- p.ix / LIST OF AWARDS AND GRANDT RECORD --- p.x / CONTENTS --- p.viii / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xv / LIST OF APPENDIXS --- p.xvi / ABBREVIATIONS --- p.xvii / Chapter CHAPTER 1 --- REVIEW OF THE LITERATURE --- p.1 / Chapter 1.1 --- What is systemic lupus erythematosus (SLE)? --- p.1 / Chapter 1.2 --- Epidemiology of SLE --- p.1 / Chapter 1.3 --- Etiology and pathogenesis of SLE --- p.3 / Chapter 1.3.1 --- Genetic factors --- p.5 / Chapter 1.3.2 --- Environmental triggers --- p.5 / Chapter 1.3.3 --- Cellular abnormalities --- p.6 / Chapter 1.3.3.1 --- APCs and cell debris clearance --- p.6 / Chapter 1.3.3.2 --- B cell and the production of autoantibodies --- p.8 / Chapter 1.3.3.3 --- T cell and the regulation of the immune responses --- p.9 / Chapter 1.3.4 --- Disturbance of the innate immune response --- p.10 / Chapter 1.3.4.1 --- PAMPs and DAPMs: all we need to know about danger in SLE --- p.10 / Chapter 1.3.4.2 --- Sensors of innate immunity --- p.12 / Chapter 1.3.4.2.1 --- TLRs sensing nucleic acids --- p.14 / Chapter 1.3.4.2.2 --- NLRs sensing bacterial peptidoglycans --- p.16 / Chapter 1.3.4.2.3 --- RAGE sensing dangerous signals --- p.16 / Chapter 1.3.5 --- Dysregulation of cytokine networks --- p.19 / Chapter 1.3.5.1 --- Anti-inflammatory cytokines --- p.20 / Chapter 1.3.5.2 --- Proinflammatory cytokines --- p.20 / Chapter 1.3.6 --- Abnormal signaling transduction --- p.22 / Chapter 1.4 --- Clinical features of SLE --- p.22 / Chapter 1.5 --- Laboratory features of SLE --- p.26 / Chapter 1.6 --- Assessing disease activity and damage of SLE --- p.28 / Chapter 1.7 --- Treatment of SLE --- p.28 / Chapter 1.7.1 --- Current immunosuppressive therapy --- p.28 / Chapter 1.7.2 --- Novel biologic therapies --- p.30 / Chapter 1.8 --- Human papillomavirus (HPV) infection in SLE --- p.32 / Chapter 1.8.1 --- Are women with lupus at higher risk of HPV infection? --- p.32 / Chapter 1.8.2 --- Abnormalities of TLR-IFN axis potentially increases HPV risk --- p.32 / Chapter 1.8.3 --- TLR suppressing mediciation potentially increases HPV risk --- p.34 / Chapter CHAPTER 2 --- HYPOTHESISS AND AIMS --- p.35 / Chapter CHAPTER 3 --- METHODOLOGIES --- p.36 / Chapter 3.1 --- Materials --- p.36 / Chapter 3.1.1 --- Selection of patients and controls --- p.36 / Chapter 3.1.2 --- Blood and cervical samples --- p.36 / Chapter 3.1.3 --- Cervical epithelial cell lines --- p.37 / Chapter 3.1.4 --- Culture medium and serum supplement --- p.37 / Chapter 3.1.5 --- Culture ligands --- p.37 / Chapter 3.1.6 --- Reagents for flow cytometric analysis (FCM) --- p.37 / Chapter 3.1.7 --- Antibodies for FCM --- p.38 / Chapter 3.1.8 --- Quantative assay kits --- p.39 / Chapter 3.1.9 --- Membrane array of phosphorylated intracellular kinases --- p.39 / Chapter 3.1.10 --- Primers for qPCR --- p.40 / Chapter 3.2 --- Methods --- p.40 / Chapter 3.2.1 --- Study design and patient assessment --- p.40 / Chapter 3.2.2 --- Isolation of PBMCs --- p.41 / Chapter 3.2.3 --- Isolation of monocytes --- p.42 / Chapter 3.2.4 --- Cell culture --- p.42 / Chapter 3.2.5 --- Sampling procedure of cervical epithelial cells --- p.42 / Chapter 3.2.6 --- HPV identification --- p.43 / Chapter 3.2.7 --- Flow cytometry gating of target cells --- p.43 / Chapter 3.2.8 --- FCM of target molecules and phosphorylated signaling molecules --- p.45 / Chapter 3.2.9 --- Membrane array of phosphorylated intracellular kinases --- p.46 / Chapter 3.2.10 --- Cytokine cytometric bead array --- p.46 / Chapter 3.2.11 --- Enzyme-linked immunosorbent assay --- p.46 / Chapter 3.2.12 --- Real-time qPCR --- p.46 / Chapter 3.2.13 --- Statistical analysis --- p.47 / Chapter CHAPTER 4 --- DOWN-REGULATED NOD2 BY IMMUNOSUPPRESSANTS IN PERIPHERAL BLOOD CELLS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS REDUCES THE MURAMYL DIPEPTIDE-INDUCED IL-10 PRODUCTION --- p.48 / Chapter 4.1 --- INTRODUCTION --- p.48 / Chapter 4.2 --- METHODS --- p.49 / Chapter 4.2.1 --- Patient selection and assessment --- p.49 / Chapter 4.2.2 --- FCM of NOD2 expression in T, B cells, monocytes and DCs --- p.49 / Chapter 4.2.3 --- Cell culture --- p.49 / Chapter 4.2.4 --- Quantitative assay --- p.49 / Chapter 4.2.5 --- Statistical analyses --- p.49 / Chapter 4.3 --- RESULTS --- p.50 / Chapter 4.3.1 --- Characteristics of lupus patients and control subjects --- p.50 / Chapter 4.3.2 --- Identification of DCs, T, B lymphocytes and monocytes --- p.50 / Chapter 4.3.3 --- Protein level of NOD2 in DCs, T, B lymphocytes and monocytes --- p.53 / Chapter 4.3.4 --- Potential explanatory variables associated with NOD2 levels in lupus patients --- p.55 / Chapter 4.3.5 --- Induction of inflammatory cytokines by NOD2 ligand --- p.61 / Chapter 4.4 --- DISCUSSION --- p.63 / Chapter 4. --- 5 CONCLUSIONS --- p.67 / Chapter CHAPTER 5 --- MEMBERS OF THE RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS AXIS AS POTENTIAL THERAPEUTIC TARGETS IN SYSTEMIC LUPUS ERYTHEMATOSUS --- p.68 / Chapter 5.1 --- INTRODUCTION --- p.68 / Chapter 5.2 --- METHODS --- p.68 / Chapter 5.2.1 --- Patients selection and assessment --- p.68 / Chapter 5.2.2 --- FCM of monocyte-surface flRAGE --- p.69 / Chapter 5.2.3 --- Cell culture --- p.69 / Chapter 5.2.4 --- Quantitative assay --- p.69 / Chapter 5.2.5 --- Membrane array of phosphorylated of intracellular kinases --- p.69 / Chapter 5.2.6 --- FCM of activated intracellular signaling molecules --- p.69 / Chapter 5.2.7 --- Statistical analysis --- p.69 / Chapter 5.3 --- RESULTS --- p.69 / Chapter 5.3.1 --- Characteristics of SLE patients --- p.69 / Chapter 5.3.3 --- Relationships between RAGE isoforms and HMGB1 --- p.75 / Chapter 5.3.4 --- Potential explanatory variables associated with levels of RAGE isoforms and HMGB1 in LN patients --- p.77 / Chapter 5.3.5 --- Activity of HMGB1 alone or in combine with TLR9 ligand --- p.81 / Chapter 5.4 --- DISCUSSION --- p.84 / Chapter 5.5 --- CONCLUSIONS --- p.88 / Chapter CHAPTER 6 --- ANTAGONIST-MEDIATED DOWN-REGULATION OF TOLL-LIKE RECEPTOR INCREASES THE PREVALENCE OF HUMAN PAPILLOMAVIRUS INFECTION IN SYSTEMIC LUPUS ERYTHEMATOSUS --- p.89 / Chapter 6.1 --- INTRODUCTION --- p.89 / Chapter 6.2 --- METHODS --- p.90 / Chapter 6.2.1 --- Patient selection and assessment --- p.90 / Chapter 6.2.2 --- HPV sampling procedure and identification --- p.90 / Chapter 6.2.3 --- FCM of TLRs 3, 7, 8 and 9 in cervical epithelial cells --- p.90 / Chapter 6.2.4 --- Cell culture --- p.90 / Chapter 6.2.5 --- Quantitative assay --- p.90 / Chapter 6.2.6 --- Real-time qPCR of Interferon-stimulated genes (ISGs) --- p.90 / Chapter 6.2.7 --- Statistical analysis --- p.91 / Chapter 6.3 --- RESULTS --- p.91 / Chapter 6.3.1 --- Pap smear findings, socio-demographic and clinical characteristics --- p.91 / Chapter 6.3.2 --- Identification of cervical epithelial cells --- p.95 / Chapter 6.3.3 --- Protein level of TLRs 3, 7, 8 and 9 in cervical epithelial cells --- p.96 / Chapter 6.3.4 --- Potential explanatory variables associated with TLR levels in lupus patients --- p.98 / Chapter 6.3.5 --- TLRs and ISGs expressions are inhibited by oncogenic HPVs --- p.102 / Chapter 6.3.6 --- Induction of inflammatory cytokines by TLR agonists was impaired in oncogenic HPVs --- p.103 / Chapter 6.4 --- DISCUSSION --- p.105 / Chapter 6.5 --- CONCLUSIONS --- p.107 / Chapter CHAPTER 7 --- CONCLUSIONS OF THE THESIS --- p.108 / Chapter 7.1 --- Answers to the hypotheses --- p.108 / Chapter 7.2 --- Conclusions and implications --- p.109 / Chapter 7.3 --- Liminations and future plan --- p.110 / Chapter 7.3.1 --- Liminations of study design --- p.110 / Chapter 7.3.2 --- Liminations of methodology --- p.111 / Chapter 7.3.3 --- Liminations of CHAPTER 4 and future plan --- p.111 / Chapter 7.3.4 --- Liminations of CHAPTER 5 and future plan --- p.112 / Chapter 7.3.5 --- Liminations of CHAPTER 6 and future plan --- p.112 / Chapter CHAPTER 8 --- REFERENCES --- p.114 / Chapter CHAPTER 9 --- APPENDIX --- p.142

Background: Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease characterised by autoantibody production and variable clinical features, ranging from mild to severe disease. Patients with SLE are at increased risk of developing accelerated atherosclerosis. Biomarkers have potential utility in SLE as markers of disease or predictors of future clinical events and mortality. Objective The aim of this thesis was to identify serological biomarkers predictive for erosive arthritis (EA), cardiovascular events (CVEs), mortality and subclinical atherosclerosis in SLE. Methods: In chapters 2 to 4, study subjects were SLE patients from Bath. Anti-cyclic citrullinated peptide antibodies (ACPA) and HLA-DR and -DQ were studied for markers of EA, and anticardiolipin (aCL) and lipoprotein profiles for markers of CVEs and mortality. In chapters 5 and 6, study subjects were women with SLE from Manchester. B-mode ultrasound scans of subjects' carotid arteries were performed at baseline and follow-up time-points to detect atherosclerotic plaque. Baseline IgG and IgM antiphospholipid (aPL) antibodies and CV risk factors were studied for markers of subclinical atherosclerosis. Clinical data collected for all studies included SLE features and auto-antibody profiles. Results: ACPA was identified as a marker of a SLE phenotype with EA - "rhupus". Patients with major erosive arthritis were HLA-DQB1*0302 carriers. Increased aCL GPL levels and total cholesterol : high density lipoprotein-C (TC : HDL-C) ratio were markers for future CVEs, and increased TC : HDL ratio, aCL GPL and lipoprotein(a) concentrations were markers for increased mortality. Lower HDL-C concentrations and anti-annexin A5 (anti-AnxA5) GPL were markers of carotid plaque progression. Conclusion: This thesis identified new markers for EA, subclinical atherosclerosis and future CVE and mortality risk in SLE. Strategies to incorporate these new CV markers into clinical CV risk assessments may assist in distinguishing the subset of SLE patients most at risk of developing accelerated atherosclerosis.

In this series of work, we examined the role of measuring cytokine mRNA expression in the urinary sediments for the assessment of SLE disease activity. The Th-1/Th-2 imbalance observed in patients with active lupus nephritis supports its relevance in pathogenesis. Our results also suggest that urinary T-BET-to-GATA-3 expression ratio may predict lupus flare. The measurement of mRNA expression in the urinary sediment may provide valuable information for the assessment and risk stratification of SLE patients. (Abstract shortened by UMI.) / In this series of work, we investigated (i) the pattern of cytokine gene expression in the urinary sediment of lupus patients, (ii) the relation between the gene expression profile in the urinary sediment and the clinical and histological disease activity of lupus patients; and (iii) the application of this non-invasive method on the assessment and monitoring of SLE disease activity. / Systemic lupus erythematosus (SLE) is a relapsing autoimmune disease with clinical manifestations that affect multiple organ systems. Lupus nephritis (LN) is recognized as one of the most severe organ involvement in SLE and affects half of the lupus patients. LN is characterized by intra-renal lymphocyte activation and inflammation. Since most of the cytokines exert their effects in a paracrine fashion, measuring their expression at the site of pathology should be of biologic relevance. Although kidney biopsy is widely used to determine the histology and severity of LN, this invasive procedure has its own risk and is not practical for serial monitoring. We hypothesize that measurement of messenger RNA (mRNA) expression in the urinary sediment may provide a non-invasive means to assess the disease activity of lupus patients. / Chan Wing Yan. / "August 2005." / Adviser: Cheuk Chun Szeto. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3692. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 302-333). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.