Erlangen-Nürnberg, Universiẗat, Diss., 2007. / Aus: Ophthalmic research ; 39.2007 u.d.T.: A suggestive association of fuchs heterochromic cyclitis with cytotoxic T cell antigen 4 gene polymorphism / Bernd M. Spriewald ...

Universiẗat, Diss., 2004--München.

Master of Science / Department of Clinical Sciences / Amy Rankin / Objective- To evaluate the use of a topical ophthalmic steroid (1% prednisolone acetate) and non-steroidal anti-inflammatory drug (0.1% diclofenac) on blood glucose concentrations, serum fructosamine concentrations, and clinical scores in diabetic dogs with cataracts using descriptive analysis. Animals- Twelve client-owned dogs with naturally-occurring, controlled (per history and physical examination), insulin-treated diabetes mellitus and cataract. A total of 40 dogs will be enrolled in the study, as determined by power analysis. Procedures- This was a prospective, randomized, double-masked, experimental study with 2 phases of data collection. Dogs were enrolled from October 2011 to March 2014 and were assigned to 1 of 2 treatments (Drug Red or Drug Blue) using blocked randomization; dogs received either 1% prednisolone acetate suspension or 0.1% diclofenac solution. Patient history, physical, and ophthalmic examinations were performed and a clinical score assigned at enrollment (Phase 1 [day 0]) and upon return (Phase 2 [day 32]). At these times, a complete blood count, serum chemistry, urinalysis, and serum fructosamine concentration were performed prior to hospitalization for up to 72 hours for continuous glucose monitoring. For 4 weeks (day 3 to 31), dogs returned home, and owners administered the dispensed ophthalmic medication 4 times daily to both eyes. Descriptive analysis of data was performed; statistical analysis will follow enrollment of 40 dogs. Results- Twelve dogs have completed the study, with 6 dogs assigned to each treatment group. Dogs received 4.44 or 0.44 mg/day of prednisolone acetate or diclofenac, respectively. Dogs assigned to Drug Red more commonly exhibited elevations in serum liver enzyme activity. Drug Red group showed a greater percent increase in fructosamine concentrations over time. Based on glucose curves alone (22 curves analyzed), an insulin dose increase was recommended for 12 curves. An insulin dose decrease and no dose change were recommended for 5 curves each. During treatment, 1 dog reportedly developed polyuria and polydipsia. Conclusions- Descriptive analysis revealed differences in some outcomes of interest among dogs treated with 2 different ophthalmic anti-inflammatory medications. Data collection is ongoing to determine if statistically significant differences exists for outcomes per group.

Neste estudo, foram descritos os aspectos clínicos e resultados visuais em indivíduos com evidência sorológica e sinais clínicos de toxoplasmose ocular. Os sujeitos foram examinados com lâmpada de fenda, exame de oftalmoscopia indireta, tendo registros fotográficos com retinografia e tomografia de coerência óptica. Duzentos e sessenta e sete participantes foram incluídos no estudo (n = 350 olhos). A forma de toxoplasmose ocular foi considerada primária em 52 indivíduos (19,5%), recorrente ativa em 89 (33,3%) e inativa em 126 (47,2%). A maioria dos olhos apresentou uma lesão (n=169; 48,3%), enquanto que 149 olhos (42,6%) apresentaram duas a quatro lesões e 32, cinco ou mais lesões (9,1%). As lesões centrais estiveram presentes em 127 olhos (36,3%), periféricas em 178 (50,9%), enquanto que lesões centrais e periféricas em 45 (12,6%). A maioria dos indivíduos apresentou sorologia para toxoplasma gondii (T. gondii) IgG + IgM- (n=245; 91,8%), enquanto que apenas 22 (8,2%) foram T. gondii IgG + IgM +. Do total de olhos afetados em que a acuidade visual foi medida (n=314), a maioria (n=160; 50,9%) apresentou melhor acuidade visual corrigida final >20/40, e 25,8% (n=81) foram considerados cegos (<20/400). Lesões múltiplas, de localização central e com tamanho maior que um diâmetro de disco óptico foram consideradas fator de risco para pior prognóstico visual. A membrana epirretiniana (n=21; 7,1%) e opacidade vítrea (n=20; 6,8%) foram as principais causas de complicações observadas. A taxa de incidência de complicações foi de 0,41 complicações/ano, e foram verificadas 0,13 reativações/ano. O estudo demonstrou altas taxas de déficit visual, devendo ser realizados novos estudos para o desenvolvimento de novas modalidades terapêuticas para diminuir o impacto da doença como causa de cegueira e deficiência visual. / In this study, we describe the clinical aspects clinical aspects and visual outcomes in individuals with serological evidence and clinical signs of ocular toxoplasmosis. The subjects were examined with a slit lamp, indirect ophthalmoscopy examination, having photographic records with retinography and optical coherence tomography. Two hundred and sixty -seven subjects were included in the study (n=350 eyes). The form of ocular toxoplasmosis was considered primary active in 52 subjects (19.5%), recurrent active in 89 subjects (33.3%) and inactive in 126 (47.2%). Most eyes presented only one lesion (n=169; 48.3%), whereas 149 individuals (42,6%) had 2-4 lesions, and 32 had five or more lesions (9.1%). Central lesions only were present in 127 eyes (36,3%), peripheral in 178 (50,9%%), while concomitant central and peripheral were present in 45 (12.6%). Most subjects had T. gondii IgG+ IgMserology (n=245; 91.8%), whereas only 22 (8,2%) were T. gondii IgG+ IgM+. From the total of affected eyes which visual acuity was measured (n=314), most (n=160; 50,9%) had better final corrected visual acuity> 20/40 and 25.8% (n = 81) were considered blind (<20/400). Multiple, centrally located lesions of greater size than an optic disc diameter were considered risk factor for a worse visual prognosis. The epiretinal membrane (n=21; 7.1%) and vitreous opacity (n=20; 6.8%) were the main causes of complications seen. The incidence rate of complications was 0,41 complications/year, and it was verified 0,13 reactivations/year. The study demonstrated a high rate of visual impairment, and studies for the development of novel therapeutic modalities should be performed to reduce the impact of the disease as a cause of blindness and visual impairment.

葡萄膜炎是一組複雜的眼內炎性疾病，可導致嚴重的視力損害，約占世界範圍內工作年齡人群組致盲眼病的10%。孫然治療上已取得一定的進步，但找尋安全有效的治療方式仍是一個臨床難題。基於解剖學分類，葡萄膜炎分為前葡萄膜炎、中葡萄膜炎、后葡萄膜炎和全葡萄膜炎。其中，前葡萄膜炎 (AU) 為最常見的臨床形式。此外，基於病因學葡萄膜炎也可被歸類為感染性和非感染性兩大亞型。作為一種炎症性疾病，已有研究表明許多內源性的免疫機制及遺傳因素參與葡萄膜炎的形成。 / 基於對葡萄膜炎疾病進程的深入瞭解，兩條主要潛在的致病通路：T細胞反應途徑和補體系統顯示在分子水平與疾病密切相關。本研究涉及參與上述兩通路的諸多因子，旨在調查葡萄膜炎的遺傳易感性，揭示潛在致病機理，以及發現新的臨床診斷標記物。 / 本研究共納入501名參與者，包括98名AU患者，95名非感染性中後葡萄膜炎 (NIPU) 患者，病例收集自香港眼科醫院及威爾斯親王醫院。此外，308名年齡50歲以上，排除了主要眼科疾患及系統性免疫疾病的健康人被招募為對照組。全面詳細記錄病人資料及臨床信息。進一步剖析葡萄膜炎疾病特點及補體通路參與程度，深入研究補體基因的累加效應及相互作用，以期發現臨床標記物。最後，採用基因型表型相關性分析，探索其與疾病嚴重程度和進展的關係。 / 研究1：系統性綜述在葡萄膜炎基因學研究上的最新發現，研究表明多種基因與葡萄膜炎疾病相關，所涉基因包括白介素、趨化因子、腫瘤壞死因子，以及參與補體和氧化途徑的相關基因。我們廣泛調查了葡萄膜炎的遺傳易感性。基因多態性選擇基於疾病的免疫及炎性特徵。（1）Interleukin與CFH基因，分別參與T細胞反應及補體通路。（2）CFB，CFH的拮抗因子，共同參與了補體旁路的調控。（3）調查C1INH(SERPING1) 因子，闡明補體經典通路在葡萄膜炎形成中的作用。（4）C3和C5基因，分別作為補體系統的“中心“因子及下游調控因子，其與葡萄膜炎的相關性被深入調查。 / 研究 2：首先探索性地調查免疫相關基因的單核苷酸多態性，包括CFH，KIAA1109 與 IL27 基因，我們的結果顯示 CFH 基因多態性 (rs800292，rs1065489) 與前葡萄膜炎顯著相關。更重要的是，CFH-rs1065489 TT基因型被確認為臨床標記物，此基因型攜帶者表現更高的葡萄膜炎復發頻率。此外，易感基因與HLA-B27的交互作用以及性別敏感性差異亦被發現。本研究的第二部份，此三個候選基因在另一個葡萄膜炎亞組NIPU中被進一步調查，CFH基因多態性 (rs800292，rs1065489) 與NIPU相關。KIAA1109-rs4505848也被發現與白塞氏病密切相關。此外，該基因多態性亦表現性別差異，較之對照組，顯性基因型頻率在男性NIPU組表現較高(GG/AG vs. AA)。 / 研究 3：CFB，作為CFH的拮抗因子，共同參與補體旁路的調控，其編碼基因被進一步調查。在AU研究中，位於C2/CFB區域的rs1048709被發現與其密切相關，該遺傳敏感性受HLA-B27影響。此外，我們還發現一個單體型 (AATA) 以及CFH與CFB的疊加效應均可導致AU風險度增高。同時，攜帶rs1048709(AG) 基因型患者傾向于更高程度的前房細胞數及KP比例。在NIPU研究中，類似的與CFB基因上的不同易感位點rs4151657相關性亦被檢測到。 / 研究 4, 5, 6: 在隨後的研究中，參與補體通路的其他三個候選基因 (SERPING1，C3和C5) 被進一步評估，儘管多種深入的分析方法被應用，但均未顯示出與疾病明確的相關性。 / 綜上，我們的結果首次揭示補體系統及其分子因素在葡萄膜炎進程中起著至關重要的作用。參與補體旁路調控的細胞因子 CFH 與 CFB，被確認為疾病風險因素。此外，分別參與補體經典通路或下游調控體系的其他三個候選基因 SERPING1(C1INH)，C3 和 C5， 以及參與T細胞反應通路的IL2_21區域和IL27基因在葡萄膜炎的發生發展中所起作用有限。因此，將來對於葡萄膜炎基因學及免疫學的研究應著眼于補體系統及其旁路途徑。 / Uveitis is a group of heterogeneous ocular inflammatory diseases with complex phenotypes, which causes substantial visual impairment and accounts for about 10% of blindness worldwide among the working age group. Despite considerable progress in treatment, safe and effective management is still a clinical challenge. Uveitis can be anatomically classified as anterior, intermediate, posterior, and panuveitis, anterior uveitis (AU) is the most common form. Based on etiology, uveitis can be also categorized as infectious and noninfectious subtypes. Uveitis is generally accepted as an inflammatory condition and regulated by various endogenous immunological mechanisms. Moreover, uveitis can occur in individuals with genetic predisposition coupled with environmental factors. / Based on our understanding of the critical checkpoints in the uveitis process, two major pathways, T-cell response and complement system, appear to be most related to uveitis in the molecular level. We therefore target on several molecular factors involved in the two major pathways to evaluate the genetic impact on susceptibility to uveitis, reveal potential disease mechanisms, and discover diagnostic markers. / We recruited a total of 501 unrelated Chinese individuals at the Hong Kong Eye Hospital and the Prince of Wales Hospital in Hong Kong, including 98 AU patients, 95 patients with noninfectious intermediate and posterior uveitis (NIPU), and 308 control subjects aged ≥ 50 years without major eye diseases or any systemic immune-related disorders. Clinical information and demographic conditions of the patients were documented. We moved on to depict the disease profile and estimate the contribution of each complement activation pathway in uveitis process. We also conducted interaction analysis among the complement factor genes to reveal the putative clinical markers for uveitis. Genotype-phenotype correlations were performed to explore their relationships with the disease severity and progression. / Study 1: In a systemic review to explore recent genetic findings in uveitis susceptibility, we found several genes persistently associated with uveitis and involved in various pathways. They are genes expressing interleukin, chemokine, tumor necrosis factor, and genes involved in complement and oxidation pathways. Genetic polymorphisms were selected based on the immunological and inflammatory properties of uveitis. (1) Interleukin and CFH genes, involving in the T-cell response and complement system respectively. (2) CFB, as a competitor of CFH, involved in the alternative pathway of complement cascade together. (3) Investigation of C1INH (SERPING1) in uveitis, with a view to elucidating the involvement of the classic pathway of complement cascade in uveitis development. (4) Evaluation of C3 and C5 genes in uveitis, due to their respective role of center component and downstream factor in the complement cascade. / Study 2: Genetic variations in the CFH, KIAA1109 and IL27 genes were examined. Our results showed an association between AU and CFH polymorphisms (rs800292 and rs1065489). The frequency of the CFH-rs800292 184G allele and GG homozygosity was higher in female patients than in controls. CFH-rs1065489 TT genotype was identified as a clinical marker associated with higher uveitis recurrence frequency. Interactions with HLA-B27 status in AU patients and different gender susceptibility were observed. In the second part of this study, these three candidate genes were examined in the other uveitic entity, NIPU, in our study cohort. CFH gene polymorphisms (rs1065489 and rs800292) were associated with NIPU patients. Specific association between KIAA1109-rs4505848 polymorphism and Behçet’s disease was identified. There was also gender specific genetic difference. The dominant genotype of KIAA1109-rs4505848 in male NIPU patients was significantly more frequent than in male controls (GG/AG vs. AA). / Study 3: CFB, a competitor of CFH and participated in the same complement alternative pathway, was investigated. SNP rs1048709 in the C2/CFB region was associated with AU, and this genetic influence was affected by HLA-B27 status. Furthermore, one haplotype block across CFB (AATA) significantly predisposed AU with an increased risk of 1.97 (95% CI: 1.41-2.76; Pcorr=0.0005). Joint effects of CFB-rs1048709 with (CFH-rs800292 and CFH-rs1065489) were identified to be at a risk of 7.48 and 7.0 respectively. In addition, patients carrying rs1048709 (AG) were predicted to develop a higher degree of anterior chamber cells and higher proportion of keratic precipitate (KP) during AU course. For NIPU, association with CFB was detected for a different SNP, rs4151657, in female patients only. / Studies 4, 5, and 6: Three candidate genes (SERPING1, C3 and C5) across the complement cascade were orderly evaluated in the whole study cohort of AU, NIPU and controls. They did not show any significant associations with both two uveitis entities, although multiple in-depth analyses have been performed. / Collectively, our results provide evidence for the involvement of the complement system in the disease pathogenesis of uveitis. CFH and CFB, involved in the complement alternative pathway, are identified as genetic risk factors for uveitis. Other complement pathway genes, SERPING1 (C1INH), C3 and C5, as well as IL2_21 region and IL27 involving in the T-cell response, confer either no or limited risk for the development of uveitis. Future genetic and immunologic investigations in uveitis should therefore be focused on the complement system and the alternative pathway. / 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. / Yang, Mingming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 148-163). / Abstracts also in Chinese. / Title page --- p.i / Abstract --- p.iii / 摘要 --- p.vii / Acknowledgement --- p.x / Table of Contents --- p.xi / List of Tables --- p.xvi / List of Figures --- p.xx / Abbreviations --- p.xxi / Publications and Conference Presentation --- p.xxiv / Awards Received --- p.xxvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Uveitis-one of the most challenging dilemmas in ophthalmology --- p.1 / Chapter 1.1.1 --- Classification of uveitis --- p.4 / Chapter 1.1.2 --- Clinical characteristics of uveitis --- p.5 / Chapter 1.1.3 --- Epidemiology of uveitis --- p.6 / Chapter 1.1.4 --- Etiology of uveitis --- p.9 / Chapter 1.1.5 --- Current management of uveitis and future perspectives --- p.11 / Chapter 1.2 --- The Descriptive Complexity of Uveitis --- p.13 / Chapter 1.3 --- Uveitis Genetics Research Strategies --- p.14 / Chapter 1.3.1 --- Candidate gene association study --- p.14 / Chapter 1.3.2 --- The identification of new genes --- p.15 / Chapter 1.4 --- Statistical Genetics for Uveitis --- p.16 / Chapter 1.4.1 --- Hardy-Weinberg equilibrium test --- p.16 / Chapter 1.4.2 --- Univariate analysis --- p.16 / Chapter 1.4.3 --- Linkage disequilibrium --- p.17 / Chapter 1.4.4 --- Haplotype analysis --- p.18 / Chapter 1.4.5 --- Multivariable analysis --- p.19 / Chapter Chapter 2 --- Objectives --- p.21 / Chapter Chapter 3 --- General Materials and Methods --- p.23 / Chapter 3.1 --- Overall Study Design --- p.23 / Chapter 3.2 --- Research Ethics --- p.23 / Chapter 3.3 --- Study Subjects Recruitment --- p.23 / Chapter 3.4 --- Demographic and Clinical Characteristics of Patients --- p.24 / Chapter 3.4.1 --- Anterior uveitis (AU) --- p.24 / Chapter 3.4.2 --- Non-infectious intermediate and posterior uveitis (NIPU) --- p.25 / Chapter 3.5 --- General Methods --- p.26 / Chapter 3.5.1 --- Total genomic DNA extraction in study subjects --- p.26 / Chapter 3.5.2 --- Taqman SNP genotyping --- p.27 / Chapter 3.5.3 --- Nested polymerase chain reaction (nPCR) --- p.27 / Chapter 3.6 --- Statistical Analysis --- p.28 / Chapter 3.6.1 --- Hardy-Weinberg equilibrium test --- p.28 / Chapter 3.6.2 --- Individual SNP association analysis --- p.28 / Chapter 3.6.3 --- Pairwise linkage disequilibrium and haplotype analysis --- p.30 / Chapter 3.6.4 --- Genotype-phenotype correlation analysis --- p.31 / Chapter 3.6.5 --- Gene-gene interaction analysis --- p.31 / Chapter Chapter 4 --- Investigation into Genetic Determinants of Uveitis --- p.32 / Chapter 4.1 --- A Critical Review on The Roles of Genetic Factors in Uveitis --- p.32 / Chapter 4.1.1 --- Human leukocyte antigens (HLA) --- p.33 / Chapter 4.1.2 --- Interleukin (IL) genes --- p.36 / Chapter 4.1.3 --- Chemokine and chemokine receptor genes --- p.37 / Chapter 4.1.4 --- Tumor necrosis factor (TNF) genes --- p.39 / Chapter 4.1.5 --- Other genes implicated in susceptibility to uveitis --- p.40 / Chapter 4.1.6 --- Complement system --- p.42 / Chapter 4.1.7 --- Conclusions and directions --- p.43 / Chapter 4.2 --- Interleukin and CFH Polymorphisms in Uveitis --- p.49 / Chapter 4.2.1 --- Introduction --- p.49 / Chapter 4.2.2 --- Study subjects --- p.50 / Chapter 4.2.3 --- SNP selection and genotyping --- p.50 / Chapter 4.2.4 --- Statistical analysis --- p.51 / Chapter 4.2.5 --- Association of interleukin and CFH polymorphisms with AU --- p.51 / Chapter 4.2.5.1 --- Association between SNPs and AU --- p.51 / Chapter 4.2.5.2 --- Association between SNPs and AU stratified by gender --- p.52 / Chapter 4.2.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.52 / Chapter 4.2.5.4 --- Genotype-phenotype correlation analysis --- p.53 / Chapter 4.2.6 --- Association of interleukin and CFH polymorphisms with NIPU --- p.53 / Chapter 4.2.6.1 --- Association between SNPs and NIPU --- p.53 / Chapter 4.2.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.54 / Chapter 4.2.6.3 --- Association between SNPs and NIPU stratified by gender --- p.54 / Chapter 4.2.7 --- Discussion --- p.55 / Chapter 4.2.7.1 --- Association of interleukin and CFH polymorphisms with AU --- p.55 / Chapter 4.2.7.2 --- Association of interleukin and CFH polymorphisms with NIPU --- p.58 / Chapter 4.3 --- C2/CFB Polymorphisms in Uveitis --- p.70 / Chapter 4.3.1 --- Introduction --- p.70 / Chapter 4.3.2 --- Study subjects --- p.70 / Chapter 4.3.3 --- SNP selection and genotyping --- p.70 / Chapter 4.3.4 --- Statistical analysis --- p.71 / Chapter 4.3.5 --- Association of C2/CFB polymorphisms with AU --- p.71 / Chapter 4.3.5.1 --- Association between SNPs and AU --- p.71 / Chapter 4.3.5.2 --- Association between SNPs and AU stratified by HLA-B27 status --- p.72 / Chapter 4.3.5.3 --- Linkage disequilibrium and haplotype association analysis --- p.73 / Chapter 4.3.5.4 --- Genotype-phenotype correlation analysis --- p.73 / Chapter 4.3.5.5 --- Joint-effect analysis between CFH and CFB in AU --- p.73 / Chapter 4.3.6 --- Association of C2/CFB polymorphisms with NIPU --- p.74 / Chapter 4.3.6.1 --- Association between SNPs and NIPU --- p.74 / Chapter 4.3.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.74 / Chapter 4.3.6.3 --- Association between SNPs and NIPU stratified by gender --- p.75 / Chapter 4.3.6.4 --- Linkage disequilibrium and haplotype association analysis --- p.75 / Chapter 4.3.7 --- Discussion --- p.75 / Chapter 4.3.7.1 --- Association of C2/CFB polymorphisms with AU --- p.75 / Chapter 4.3.7.2 --- Association of C2/CFB polymorphisms with NIPU --- p.77 / Chapter 4.4 --- SERPING1 Gene Polymorphisms in Uveitis --- p.94 / Chapter 4.4.1 --- Introduction --- p.94 / Chapter 4.4.2 --- Study subjects --- p.94 / Chapter 4.4.3 --- SNP selection and genotyping --- p.95 / Chapter 4.4.4 --- Statistical analysis --- p.95 / Chapter 4.4.5 --- Association of SERPING1 polymorphisms with AU --- p.95 / Chapter 4.4.5.1 --- Association between SNPs and AU --- p.95 / Chapter 4.4.5.2 --- Association between SNPs and AU stratified by gender --- p.96 / Chapter 4.4.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.96 / Chapter 4.4.5.4 --- Association between SNPs and AU stratified by clinical features --- p.96 / Chapter 4.4.6 --- Association of SERPING1 polymorphisms with NIPU --- p.96 / Chapter 4.4.6.1 --- Association between SNPs and NIPU --- p.97 / Chapter 4.4.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.97 / Chapter 4.4.6.3 --- Association between SNPs and NIPU stratified by gender --- p.97 / Chapter 4.4.7 --- Discussion --- p.98 / Chapter 4.5 --- C3 Gene Polymorphisms in Uveitis --- p.109 / Chapter 4.5.1 --- Introduction --- p.109 / Chapter 4.5.2 --- Study subjects --- p.109 / Chapter 4.5.3 --- SNP selection and genotyping --- p.110 / Chapter 4.5.4 --- Statistical analysis --- p.110 / Chapter 4.5.5 --- Association of C3 polymorphisms with AU --- p.110 / Chapter 4.5.5.1 --- Association between SNPs and AU --- p.110 / Chapter 4.5.5.2 --- Association between SNPs and AU stratified by gender --- p.111 / Chapter 4.5.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.111 / Chapter 4.5.5.4 --- Association between SNPs and AU stratified by clinical features --- p.111 / Chapter 4.5.6 --- Association of C3 polymorphisms with NIPU --- p.111 / Chapter 4.5.6.1 --- Association between SNPs and NIPU --- p.111 / Chapter 4.5.6.2 --- Association between SNPs and NIPU stratified by subtypes and gender --- p.112 / Chapter 4.5.7 --- Discussion --- p.112 / Chapter 4.6 --- C5 Gene Polymorphisms in Uveitis --- p.126 / Chapter 4.6.1 --- Introduction --- p.126 / Chapter 4.6.2 --- Study subjects --- p.126 / Chapter 4.6.3 --- SNP selection and genotyping --- p.127 / Chapter 4.6.4 --- Statistical analysis --- p.127 / Chapter 4.6.5 --- Association of C5 polymorphisms with AU --- p.127 / Chapter 4.6.5.1 --- Association between SNPs and AU --- p.127 / Chapter 4.6.5.2 --- Association between SNPs and AU stratified by gender --- p.128 / Chapter 4.6.5.3 --- Association between SNPs and AU stratified by HLA-B27 status and clinical features --- p.128 / Chapter 4.6.6 --- Association of C5 polymorphisms with NIPU --- p.128 / Chapter 4.6.6.1 --- Association between SNPs and NIPU --- p.128 / Chapter 4.6.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.129 / Chapter 4.6.6.3 --- Association between SNPs and NIPU stratified by gender --- p.129 / Chapter 4.6.7 --- Discussion --- p.129 / Chapter Chapter 5 --- Conclusions and Future Perspectives --- p.143 / Chapter 5.1 --- General Conclusion --- p.143 / Chapter 5.2 --- Future Research in Uveitis Molecular Genetics --- p.144 / REFERENCES --- p.148

The primary aim of this research is to test the therapeutic potential of certain new generation corticosteroid drugs in order to develop safe and effective treatment for eye diseases that result in oedema, or swelling. The rising incidence of diabetes and the ageing population of developed countries mean that the prevalence of uveitis, diabetic retinopathy and age related macular degeneration will rise. Often, oedema is one of the reasons for vision loss. Corticosteroids are often used to reduce inflammation. Inflammation is one of several sources of oedema. Glucocorticoids, a class of corticosteroids that have anti-inflammatory properties, are thus used to treat ocular oedema. There is an unmet need to support clinical experience of the efficacy of steroids for ocular inflammation and oedema with more substantial scientific evidence. None of the drugs under investigation, with the exceptions of dexamethasone and triamcinolone, have been used for any ocular therapeutic purpose before. This thesis investigates “repurposing” fludrocortisone to the ophthalmic area. 11-Desoxycorticosterone (11D) and Deoxycorticosterone (DCS), other potentially valuable mineralocorticoids, remain completely untested. Lastly, Kenacort ®, or triamcinolone acetonide (TCA), is only used off-label by ophthalmologists. Methods: In the first study, corticosteroids, and especially mineralocorticoids, were investigated for their treatment efficacy in experimental uveitis, or intraocular inflammation (using a model known as endotoxin induced uveitis). In the second study, endothelial cells from choroidal blood vessels in the back of the eye were used in vitro to study whether corticosteroids reduce paracellular (between cells) permeability. Lastly, since endophthalmitis due to frequent injections is a side effect of corticosteroid use, the pharmacokinetics of different size formulations of corticosteroids were studied in an effort to find a formula that would have a prolonged dwell time within the eye.

Para elucidar alguns aspectos da imunopatogenia da uveíte na erliquiose canina, avaliaram-se, por meio da análise imunistoquímica, bulbos oculares de cães experimentalmente infectados por Ehrlichia canis (grupo 1- G1), naturalmente infectados por Ehrlichia canis (grupo 2-G2) e na co-infecção natural de E. canis e Babesia sp. (grupo 3). Parâmetros clínicos e hematológicos foram avaliados. Empregaram-se o dot-blot-Elisa e a reação de imunofluorescência indireta para E. canis e Babesia sp. respectivamente. Para a confirmação diagnóstica, utilizou-se a reação de cadeia de polimerase (PCR) para E.canis. A contagem imunofenotípica para os anticorpos CD3, CD4, CD8, Tal1B5 e MAC 387 não demonstrou diferença significativa nas diferentes regiões analisadas do bulbo ocular. Observou-se no G1, G2 e G3, em todas as regiões analisadas diferença significativa da contagem imunofenotípica de células CD8+ em relação às células CD4+. Evidenciou-se diferença significativa entre a contagem percentual de células IgG2+ e CD79?+ na região de corpo ciliar do G3 em relação ao G1. A região da íris do G3, em relação ao G2, demonstrou diferenças significativas para o anticorpo IgG1. Evidenciou-se nos três grupos, a existência de correlação linear entre as células CD3+ e CD8+ e entre as células IgG2+ e CD79?+ em diversas regiões do bulbo ocular. O infiltrado inflamatório mostrou-se mais intenso nas regiões de corpo ciliar e ângulo iridocorneal, moderado em limbo e íris e mínimo em coróide. A avaliação semiquantitativa por score da intensidade do infiltrado inflamatório mostrou-se mais intensa nos animais que apresentavam co-infecção, sugerindo uma resposta imune mais intensa nesses cães. Demonstrou-se que o infiltrado inflamatório era composto, predominantemente, por linfócitos T CD3+ e B CD79+?. A maior porcentagem de células T CD3+ era CD8+, caracterizando, portanto, uma resposta imune do tipo citotóxica. A presença de células B CD79+? fala a favor de produção local de anticorpos. Observaram-se células imunomarcadas por IgG2 e poucas células marcadas por IgG1, sugerindo uma polarização da resposta imune para o padrão Th1, sendo um possível mecanismo imune de lesão na uveíte. Observou-se alta expressão de moléculas de MHC-classe II, sugerindo uma resposta imune intensa nos tecidos oculares, contudo ineficiente devido a deficiência de células CD4+. / The immunopathogenicity of uveitis in the canine ehrlichiosis was studied by conducting anatomy and immunohistochemical analyses in the ocular globes of dogs experimentally (Group 1) and naturally (Group 2) infected with Ehrlichia canis, and naturally coinfected with Ehrlichia canis and Babesia sp. (Group 3). Clinical and hematological parameters were evaluated. Dot-blot Elisa and indirect immunofluorescent test (IFA) were used to analyze E. canis and Babesia sp., respectively. PCR assay confirmed the diagnosis of the disease caused by E. canis. The immunophenotypic analysis with the antibodies CD3, CD4, CD8, Tal1B5 and MAC 387 revealed no significant differences between the various ocular regions analyzed. Significant differences were observed between the immunophenotypic analysis of CD8+ and CD4+cells from all regions analyzed from G1, G2 and G3; between the percentile counts of IgG2+ and CD79?+ in the ciliary body of G3 dogs when compared to G1, and for the IgG1 antibody counts of the iris region from G3 when compared to G2. A linear correlation between CD3+ and CD8+ cells and between IgG2+ and CD79?+ cells from several regions of the ocular globe was found for all three groups. The cellular inflammatory infiltrate observed in the ocular tissue was severe in the regions of the ciliary bodies and iridocorneal angle, moderate in the limbus and iris, and only slightly present in the choroids. A semiquantitative analysis of the intensity of the inflammatory infiltrate of the ocular globes was more intense in G3, which suggested a greater response of the immune system in these animals. The inflammatory infiltrate was composed of mainly B CD79+? and T CD3+ lymphocytes, which had CD8+ at a high percentage, thus characterizing this as a cytotoxic immune response. Results indicated that B CD79+? cells favored the production of local antibodies. IgG2 and very few IgG1 immunolabeled cells were found. This result indicated a polarization of the immune response to the Th1 pattern, which could be the mechanism of the lesions in the uveitis. A large number of cells expressing the MHC-class II molecules were observed, suggesting an intense immune response in the ocular tissues, however ineffective due to the CD4+ cell´s deficient.

Orientador: Sonia Maria Oliani / Coorientador: Cristiane Damas Gil / Banca: Janetti Nogueira de Francischi / Banca: José Roberto Mineo / Banca: Dorotéia Rossi Silva Souza / Banca: Ana Elizabete Silva / Resumo: A proteína anexina A1 (AnxA1) apresenta importantes propriedades anti-inflamatórias e estudos sugerem que suas ações podem ser mediadas por receptores para peptídeos formilados (FPR). Embora os efeitos anti-inflamatórios da AnxA1 e de seus peptídeos miméticos, especialmente o Ac2-26, tenham sido explorados em diversas investigações, são raros os estudos da AnxA1 nas inflamações oculares. Em razão dos graves efeitos colaterais dos tratamentos atuais para a uveíte, uma importante causa de cegueira, investigamos, in vivo, os efeitos e o mecanismo de ação da AnxA1 nos tecidos oculares de roedores na uveíte induzida por endotoxina (EIU). Ratos machos (Rattus novergicus) foram anestesiados e inoculados, por via subcutânea, na pata direita com lipopolissacarídeo (LPS) (100µg) para o desenvolvimento da uveíte. Após, foram divididos em grupos experimentais (n=10/grupo): EIU por 24 e 48h; EIU por 24h e tratados farmacologicamente por administrações tópica e sistêmica do peptídeo Ac2-26 (100µg) e EIU 24h tratado sistemicamente com peptídeo e Boc2, antagonista do FPR (50µg/animal). Para confirmar a importância da AnxA1 endógena na resolução da inflamação ocular, camundongos selvagens e deficientes para AnxA1 (AnxA1-/-) foram induzidos à uveíte por 24h sem tratamento. Nesses animais AnxA1-/- a resposta inflamatória foi exacerbada em comparação com os selvagens. Enquanto, nos olhos dos ratos, as análises quantitativas dos leucócitos, dosagens de interleucina (IL)-1β, IL-6, fator de necrose tumoral (TNF)-α, óxido nítrico (NO) e expressão da ciclo-oxigenase (COX)-2 nos tecidos e/ou no humor aquoso indicaram os efeitos anti-inflamatórios do peptídeo. Efeitos que foram revertidos na presença do Boc2. As análises imuno-histoquímicas das proteínas AnxA1, AnxA1 fosforilada em... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Annexin A1 (AnxA1) is a protein that displays anti-inflammatory properties and some studies suggest that its effects may be mediated by formyl peptide receptors (FPR). Although the anti-inflammatory activities of AnxA1 and its mimetic peptides, including Ac2-26, have been explored in several investigations, the role of AnxA1 in ocular inflammatory processes has not yet been elucidated. Given the common side effects of the current therapies used to treat uveitis, an important cause of blindness worldwide, we investigated, in vivo, the AnxA1 effects and mechanism of action in endotoxin-induced uveitis (EIU). Rattus norvegicus were induced to uveitis (lipopolysaccharide - 100 µg) and divided into experimental groups (n=10/group): EIU untreated for 24 and 48h, EIU treated with topical applications (4/4h) or a single intravenous injection of Ac2-26 (100µg) and EIU systemically treated with the peptide and Boc2, the FPR antagonist (50µg/animal). To confirm the importance of endogenous AnxA1 in the resolution of ocular inflammation, wild-type and AnxA1 deficient (AnxA1-/-) mice were also induced to uveitis without treatment for 24h. AnxA1-/- mice showed exacerbated inflammation compared to wild-type animals. As, quantitative analyses of leukocytes, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, nitric oxide (NO) levels and cyclooxigenase (COX)-2 expression in ocular tissues and/or in aqueous humor of rats eyes revealed the anti-inflammatory effects of the peptide, which were abrogated in the Boc2 presence. Immunohistochemical analysis of AnxA1, serine-or-tyrosine-phosphorylated AnxA1 (AnxA-S27-PO4 - AnxA-Y21-PO4) in the ocular tissues showed AnxA1 and AnxA1-S27-PO4 expression in epithelial (cornea, iris and ciliary processes) and nervous cells. These expressions were increased in... (Complete abstract click electronic access below) / Doutor

Para elucidar alguns aspectos da imunopatogenia da uveíte na erliquiose canina, avaliaram-se, por meio da análise imunistoquímica, bulbos oculares de cães experimentalmente infectados por Ehrlichia canis (grupo 1- G1), naturalmente infectados por Ehrlichia canis (grupo 2-G2) e na co-infecção natural de E. canis e Babesia sp. (grupo 3). Parâmetros clínicos e hematológicos foram avaliados. Empregaram-se o dot-blot-Elisa e a reação de imunofluorescência indireta para E. canis e Babesia sp. respectivamente. Para a confirmação diagnóstica, utilizou-se a reação de cadeia de polimerase (PCR) para E.canis. A contagem imunofenotípica para os anticorpos CD3, CD4, CD8, Tal1B5 e MAC 387 não demonstrou diferença significativa nas diferentes regiões analisadas do bulbo ocular. Observou-se no G1, G2 e G3, em todas as regiões analisadas diferença significativa da contagem imunofenotípica de células CD8+ em relação às células CD4+. Evidenciou-se diferença significativa entre a contagem percentual de células IgG2+ e CD79?+ na região de corpo ciliar do G3 em relação ao G1. A região da íris do G3, em relação ao G2, demonstrou diferenças significativas para o anticorpo IgG1. Evidenciou-se nos três grupos, a existência de correlação linear entre as células CD3+ e CD8+ e entre as células IgG2+ e CD79?+ em diversas regiões do bulbo ocular. O infiltrado inflamatório mostrou-se mais intenso nas regiões de corpo ciliar e ângulo iridocorneal, moderado em limbo e íris e mínimo em coróide. A avaliação semiquantitativa por score da intensidade do infiltrado inflamatório mostrou-se mais intensa nos animais que apresentavam co-infecção, sugerindo uma resposta imune mais intensa nesses cães. Demonstrou-se que o infiltrado inflamatório era composto, predominantemente, por linfócitos T CD3+ e B CD79+?. A maior porcentagem de células T CD3+ era CD8+, caracterizando, portanto, uma resposta imune do tipo citotóxica. A presença de células B CD79+? fala a favor de produção local de anticorpos. Observaram-se células imunomarcadas por IgG2 e poucas células marcadas por IgG1, sugerindo uma polarização da resposta imune para o padrão Th1, sendo um possível mecanismo imune de lesão na uveíte. Observou-se alta expressão de moléculas de MHC-classe II, sugerindo uma resposta imune intensa nos tecidos oculares, contudo ineficiente devido a deficiência de células CD4+. / The immunopathogenicity of uveitis in the canine ehrlichiosis was studied by conducting anatomy and immunohistochemical analyses in the ocular globes of dogs experimentally (Group 1) and naturally (Group 2) infected with Ehrlichia canis, and naturally coinfected with Ehrlichia canis and Babesia sp. (Group 3). Clinical and hematological parameters were evaluated. Dot-blot Elisa and indirect immunofluorescent test (IFA) were used to analyze E. canis and Babesia sp., respectively. PCR assay confirmed the diagnosis of the disease caused by E. canis. The immunophenotypic analysis with the antibodies CD3, CD4, CD8, Tal1B5 and MAC 387 revealed no significant differences between the various ocular regions analyzed. Significant differences were observed between the immunophenotypic analysis of CD8+ and CD4+cells from all regions analyzed from G1, G2 and G3; between the percentile counts of IgG2+ and CD79?+ in the ciliary body of G3 dogs when compared to G1, and for the IgG1 antibody counts of the iris region from G3 when compared to G2. A linear correlation between CD3+ and CD8+ cells and between IgG2+ and CD79?+ cells from several regions of the ocular globe was found for all three groups. The cellular inflammatory infiltrate observed in the ocular tissue was severe in the regions of the ciliary bodies and iridocorneal angle, moderate in the limbus and iris, and only slightly present in the choroids. A semiquantitative analysis of the intensity of the inflammatory infiltrate of the ocular globes was more intense in G3, which suggested a greater response of the immune system in these animals. The inflammatory infiltrate was composed of mainly B CD79+? and T CD3+ lymphocytes, which had CD8+ at a high percentage, thus characterizing this as a cytotoxic immune response. Results indicated that B CD79+? cells favored the production of local antibodies. IgG2 and very few IgG1 immunolabeled cells were found. This result indicated a polarization of the immune response to the Th1 pattern, which could be the mechanism of the lesions in the uveitis. A large number of cells expressing the MHC-class II molecules were observed, suggesting an intense immune response in the ocular tissues, however ineffective due to the CD4+ cell´s deficient.

La uveítis asociada a la AIJ oligoarticular engloba a prácticamente el 75% de las uveítis anteriores en la edad pediátrica y es la enfermedad sistémica que con mayor frecuencia se encuentra asociada a uveítis en esta edad. Su prevalencia está comprendida entre un 13 y un 45%, variando en función de factores geográficos, estructuras de los centros y las vías de referencia de los pacientes. La uveítis es más frecuente en niñas y en los pacientes que presentan ANA positivo con un título superior a 1/8075. Los ANA son actualmente los únicos marcadores de riesgo biológico de la uveítis y entre un 18-45% de los pacientes con AIJ oligoarticular y ANA positivo presentan uveítis.El pronóstico de la enfermedad ocular ha mejorado de forma significativa en los últimos años debido a la puesta en marcha de los programas de cribaje precoz y a los avances en el tratamiento médico y quirúrgico. No obstante, las complicaciones se presentan hasta en un 20-40% de los pacientes y pueden ocasionar un déficit severo de la agudeza visual. En series como la de Paroli et al., un 90,5% de los ojos presentan complicaciones (64% catarata y 25% glaucoma) y Thorne et al., en un 65% en el diagnóstico inicial de la uveítis. Aunque esta tasa de complicaciones estará en relación al tipo de centro, en estudios epidemiológicos también se eleva hasta un 45% Al mismo tiempo, en series recientes publicadas con seguimiento a largo plazo la pérdida severa de agudeza visual, definida como la agudeza visual inferior a 20/200, se presenta en un 15% de los pacientes y ceguera como mínimo en un ojo después de un año de tratamiento agresivo sistémico en un 6-25% de los pacientes, siendo estos porcentajes mucho más elevados que en otros tipos de uveítis. Las secuelas visuales se producen por lo general debido a un retraso en el diagnóstico y por tratamiento tardío e inadecuado.En cuanto al manejo terapéutico, Foster et al. han comprobado que la instauración de tratamiento precoz y agresivo, en las formas más graves de uveítis, especialmente con los nuevos fármacos modificadores de la respuesta biológica, de acción antiTNF-&#945; puede mejorar el pronóstico visual a largo plazo.Este tipo de abordaje terapéutico es una tendencia en el tratamiento de las enfermedades inflamatorias en la actualidad: instaurar tratamientos más selectivos de entrada en lugar de hacer un tratamiento escalonado ya que el tratamiento inicial puede marcar el pronóstico visual final. La uveítis asociada a AIJ, al ser una enfermedad mediada por el sistema inmune, se caracteriza por la infiltración celular por células T y el daño tisular consiguiente.El curso clínico de la uveítis asociada a la AIJ, en consecuencia, puede estar influenciado por el balance entre citoquinas proinflamatorias y supresoras de lainflamación. Los niveles de producción de citoquinas por las células en cada individuo se han asociado con polimorfismos en los genes promotores de las citoquinas. Debido a que los niveles de la expresión de los genes de las citoquinas están determinados parcialmente a través de los SNP, aquéllos que afectan a la secuencia codificante o reguladora del gen pueden producir cambios importantes en la estructura de la proteína o en el mecanismo de regulación de la expresión. Todo ello, puede influenciar en la susceptibilidad a desarrollar la enfermedad inflamatoria y su severidad. Por otra parte, el estudio de determinados polimorfismos genéticos en genes inflamatorios podría permitir un avance en el conocimiento de la respuesta a los diferentes fármacos, especialmente los de acción antiTNF-&#945;.En consecuencia, el estudio de investigación de los polimorfismos de citoquinas puede ser útil para profundizar en nuevos métodos y determinar factores predictores que permitan identificar precozmente tanto el riesgo de desarrollar uveítis en la AIJ oligoarticular como la severidad de ésta. Estos factores podrían a su vez permitir individualizar el tratamiento en función del riesgo potencial de pérdida de agudeza visual y profundizar en el mejor entendimiento de la etiopatogenia de la enfermedad.A la hora de desarrollar el trabajo en que se ha basado la presente tesis doctoral, nuestras hipótesis de partida han sido:1. Los pacientes con uveítis asociada a AIJ oligoarticular presentan una mayor prevalencia de polimorfismos de genes proinflamatorios en relación a los pacientes con AIJ oligoarticular que no presentan uveítis.2. La presencia de polimorfismos de citoquinas inflamatorias se asocia a la aparición de formas más severas de uveítis en pacientes con AIJ oligoarticular.Por tanto, el objetivo principal de este estudio es el de analizar el papel de los polimorfismos de las citoquinas pro y antiinflamatorias en la presentación clínica y severidad de la uveítis en pacientes con AIJ oligoarticular. Para abordar dicho objetivo hemos establecido los siguientes apartados:1. Determinar la asociación de los polimorfismos genéticos de 5 citoquinas pro y antiinflamatorias: TNF-&#945;, IL-6, IL-1&#946;, IL-10 e IL-1Ra en este grupo de pacientes en relación a la susceptibilidad y severidad de la uveítis asociada a la AIJ oligoarticular añadiendo a las técnicas habituales el estudio de polimorfismos de genes inflamatorios.2. Establecer un perfil de riesgo con variables demográficas, oftalmológicas, reumatológicas y biológicas con el objetivo de poder identificar de manera precoz los pacientes con mayor susceptibilidad de presentar uveítis en la AIJ oligoarticular.