Made available in DSpace on 2015-12-06T23:46:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2014 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Objetivos: Comparar os valores de densidade optica de pigmento macular (MPOD) obtidos atraves dos metodos de fotometria de flicker heterocromatico (HFP) e autofluorescencia (AF) em um grupo de pacientes normais; comparar os valores de MPOD em pacientes diabeticos e nao-diabeticos, investigar a relacao entre esses valores e os niveis sericos de hemoglobina glicosilada e perfil lipidico; e, finalmente, investigar a relacao entre a idade e a distribuicao dos valores de MPOD ao redor da fovea em pacientes normais. Metodos: No primeiro estudo, 10 pacientes (20 olhos) normais (sem doencas oculares ou sistemicas) foram incluidos e testados para MPOD atraves dos metodos de HFP e AF. O metodo de AF utiliza dois comprimentos de onda diferentes para a aquisicao das imagens atraves de um oftalmoscopio de varredura a laser confocal modificado (Heidelberg Retina Angiograph, HRA, Heidelberg Engineering, Inc., Heidelberg, Alemanha). Os valores para as duas tecnicas foram obtidos em 4 excentricidades diferentes ao redor do centro da fovea (0,25, 0,5, 1 e 1,75°). Cada olho foi testado tres vezes para cada um dos metodos, e a analise estatistica foi realizada atraves de regressao linear e teste t pareado. Em um segundo estudo, 43 pacientes (43 olhos) diabeticos tipo 2 e normais foram incluidos prospectivamente e alocados em 3 grupos: grupo 1 (controle; n=14), grupo 2 (diabeticos sem sinais de retinopatia; n=17) e grupo 3 (diabeticos com retinopatia nao proliferativa leve; n=12). Todos os pacientes foram submetidos a exame oftalmologico completo e foram coletadas informacoes sistemicas e oculares, incluindo o uso de suplementos vitaminicos contendo carotenoides. A avaliacao da MPOD foi realizada atraves do HRA. Os valores obtidos em 2 excentricidades ao redor do centro da fovea (0,5 e 2°) foram comparados entre os grupos atraves da analise de variancia. Os niveis sericos de hemoglobina glicosilada e lipideos (HDL, LDL, colesterol total e triglicerides) foram correlacionados com os niveis da MPOD para cada grupo atraves de regressao linear. Finalmente, em um terceiro estudo, os valores da MPOD de 30 pacientes (30 olhos) normais foram obtidos atraves do HRA em 3 excentricidades retinianas (0,5, 1 e 2°) e correlacionados com a idade atraves de regressao linear. Resultados: Para a comparacao entre as tecnicas, os valores obtidos pela HFP foram consistente e significativamente inferiores aos valores obtidos pelo HRA (p<0,001) em todas as excentricidades retinianas testadas. Houve uma correlacao significativa entre os valores obtidos pelos dois metodos em quase todos os locais testados, sendo que a correlacao mais forte foi observada no ponto mais afastado da fovea (1,75°) (r=0,73). Em relacao a comparacao entre diabeticos e normais, a media dos valores da MPOD obtidos a 2° do centro da fovea variou significativamente para os tres grupos [grupo 1 (0,29 ± 0,07 DU), grupo 2 (0,22 ± 0,09 DU) e grupo 3 (0,14 ± 0,05 DU), p<0,001]. Alem disso, observou-se uma correlacao inversa e significativa entre os niveis de hemoglobina glicosilada e os valores medios da MPOD a 2° para todos os pacientes (r=-0,63, p<0,001). Nao foram encontradas correlacoes significativas com niveis de lipideos sericos, duracao do diabetes ou idade. Os pacientes incluidos para a analise de correlacao com a idade apresentaram 23 a 77 anos (idade media ± DP = 48,6 ± 16,4 anos). Diferencas significativas para os valores medios de MPOD obtidos a 0,5, 1 e 2° do centro da fovea foram encontradas (0,49 ± 0,12 DU; 0,37 ± 0,11 DU; 0,13 ± 0,05 DU, respectivamente, p<0,05). Os valores da MPOD a 0,5 e 1° e a idade mostraram uma correlacao significativa (p&#8804;0,02), ja os valores da MPOD a 2° nao apresentaram correlacao com a idade (p=0,06). Conclusoes: Os valores da MPOD obtidos atraves do HRA mostraram correlacao significativa com os valores obtidos pelo metodo padrao de HPF, porem foram consistentemente mais elevados em todas as excentricidades retinianas testadas. Estes resultados sugerem que o metodo pode ser usado com seguranca em pacientes incapazes de realizar o teste de HPF, o que e essencial para uma aplicacao clinica mais ampla da analise de pigmento macular. Pacientes diabeticos tipo 2 com e sem retinopatia apresentaram niveis reduzidos da MPOD quando comparados aos pacientes nao-diabeticos. Alem disso, observou-se uma correlacao inversa e significativa entre os niveis de hemoglobina glicosilada e esses valores quando todos os pacientes foram analisados. Finalmente, os valores da MPOD obtidos em uma populacao normal foram mais elevados proximo ao centro da fovea. Esses valores apresentaram pico entre 45-50 anos de idade, seguido por uma reducao gradual apos 60 anos de idade / BV UNIFESP: Teses e dissertações

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The Y402H polymorphism in complement factor H (CFH) is a common and important risk factor, where CFH is an inhibitor of the alternative complement pathway. The disease-associated protein variant (CFH402H) binds poorly to aged human macular Bruch’s membrane (BM), a site of AMD formation. Heparan sulphate (HS) is the major binding site for CFH in this extracellular matrix. Unlike CFH402Y, CFH402H binds poorly to lowly sulphated HS. The aim of this research was to investigate the presence and distribution of proteoglycan (PG) core proteins and glycosaminoglycans (GAGs) in the normal adult human macula, and to analyse potential changes with age in the quantity and composition of HS and other potential molecular determinants of disease in BM. Post mortem human eye tissue was obtained from consenting donors (age range 18-93 years), and either dissected into tissue layers or used to produce frozen macular tissue sections. Proteomic analysis of different retinal tissue layers was performed by tandem mass spectrometry. Immunofluorescence microscopy was undertaken on the macular tissue sections. Compositional analysis of HS in BM was performed by 2-aminoacridone labelling of HS disaccharides and reverse phase high performance liquid chromatography against reference HS disaccharide standards. PG core proteins were identified in BM and other macular tissue layers, including members of the basement membrane, hyalectan and short leucine-rich repeat PG families. HS, chondroitin sulphate, dermatan sulphate and hyaluronan were present throughout the retina and choroid, but keratan sulphate only in the sclera. The mean quantity of HS in BM was 47% lower (p=0.006) in old donors (n=13, 64-92 years), compared to young donors (n=6; 26-39 years). The mean level of HS sulphation was also lower in old donors, e.g. 34% vs. 39% (p=0.02) N-sulphated HS. The mean level of HS in macular BM by immunohistochemistry was approximately 50% lower (p=0.02) in old donors (n=10, 18-93 years), and the mean level of the HS PG core protein perlecan was reduced by 85% (p=0.01; n=18, 27-90 years). High levels of complement activation (C3b and membrane attack complex) were observed in some young donors. Reduced HS was associated with increased complement activation in some donors (r2 0.30). A combination of proteomics and immunohistochemistry approaches has provided the first comprehensive analysis of the presence and distribution of PG core proteins and their associated GAG chains throughout the macular layers of the normal adult human retina. These demonstrate a differential distribution according to PG core protein, GAG class and GAG sulphation state. The quantity of HS decreases substantially with age in human BM, and its sulphation level also decreases. The presence of less HS in old BM would make fewer binding sites available for CFH, and could contribute to AMD pathogenesis through increased complement activation. This idea is supported by the observation that reduced HS is associated in some individuals with increased C3b in BM. These findings have important implications for unravelling mechanisms of ocular disease and planning novel therapeutic strategies, particularly in the case of AMD.

Le trou maculaire (TM) est un défaut dans la rétine au niveau de la zone centrale de la macula (fovéa). Typiquement, les patients atteints de cette pathologie auront une diminution de leur acuité visuelle (AV) et de la métamorphopsie. Le traitement pour ces patients consistera en une vitrectomie par la pars plana (VPP) dans laquelle le vitré est retiré et remplacé par une tamponnade (SF6, C3F8, air ou huile de silicone). Après cette chirurgie, le succès chirurgical, soit de fermeture anatomique du TM, est très élevé. Toutefois, plus récemment, on s'aperçoit que malgré un taux de fermeture anatomique élevé, les résultats visuels après l'intervention chirurgicale ne sont pas aussi importants, d'où la nécessité d'identifier les facteurs pronostiques liés à la VPP dans les TM qui ont fermé chirurgicalement et de tenter de prédire les résultats visuels suivant la VPP. En ce qui concerne les TM qui n'ont pas fermé lors d'une première VPP, il est intéressant d'évaluer les résultats fonctionnels et anatomiques à la suite d'une VPP de reprise. Nos travaux ont démontré que les yeux avec une durée du TM plus courte, une taille du TM plus petite et une AV préopératoire plus élevée ont obtenu de meilleurs résultats visuels après une première chirurgie réussie. Nous avons par la suite produit un modèle hybride basé sur des données cliniques et des tomographies par cohérence optique haute définition (HD-OCT) préopératoires de TM, et ce, à l'aide de l'intelligence artificielle, afin de tenter de mieux prédire les résultats visuels. Les modèles basés sur les données cliniques ou les HD-OCT ont obtenu de bonnes performances discriminantes individuellement. Toutefois, la combinaison des deux modèles dans un modèle hybride n'a pas significativement amélioré les performances. Enfin, les TM qui subissaient une VPP de révision (non-fermeture chirurgicale à la première VPP) montraient une fermeture anatomique dans 85% des cas et avaient une amélioration de l'AV au fil du temps. / Idiopathic full thickness macular hole (MH) is a defect of all layers of the fovea. Typically, patients with this condition will experiment decreased visual acuity (VA) and metamorphopsia. Treatment for these patients will consist in a pars plana vitrectomy in which the vitreous is removed and replaced by a tamponade (SF6, C3F8, air or silicone oil). After this surgery, the surgical success, or anatomical closure of the MH, is very high. However, more recently, it has been noticed that despite a high anatomical closure rate, the visual results after the surgery are not quite as important, hence the need to identify the prognostic factors related to vitrectomy in patients with closed MH and to predict visual results following vitrectomy. For MH that did not close during the primary vitrectomy, it is interesting to assess the functional and anatomical results following a revision vitrectomy. Our works provided that eyes with shorter MH duration, smaller MH size and higher preoperative VA achieved better visual outcomes after successful MH surgery. We then produced a hybrid model based on clinical data and preoperative MH high-definition optical coherence tomography (HD-OCT), using artificial intelligence to try to better predict visual results. Both the clinical data and HD-OCT models had good discriminative performances. Combining both into a hybrid model did not significantly improve performance. Finally, MH that underwent revision vitrectomy (nonsurgical closure at primary vitrectomy) showed anatomical closure in 85% of cases and had VA improvement over the time.

No / To evaluate repeatability and reproducibility of macular thickness measurements in visually normal eyes using the Topcon 3D OCT-1000. Methods: Phase 1 investigated scan repeatability, the effect of age and pupil dilation. Two groups (6 younger and 6 older participants) had one eye scanned 5 times pre and post- dilation by 1 operator. Phase 2 investigated between-operator, within and between-visit reproducibility. 10 participants had 1 un-dilated eye scanned 3 times on 2 separate visits by 2 operators. Results: Phase 1: No significant difference existed between repeat scans (p=0.75) and no significant difference was found pre- and post-dilation (p=0.54). In the younger group variation was low (95% limits ± 3.62 m) and comparable across all retinal regions. The older group demonstrated greater variation (95% limits ± 7.6 m). Phase 2: For a given retinal location, 95% confidence limits for within-operator, within-visit reproducibility was 5.16 m. This value increased to 5.56 m for the same operator over two visits and to 6.18 m for two operators over two visits. Conclusion: A high level repeatability, close to 6 m, of macular thickness measurement is possible using the 3D OCT- 1000. Measured differences in macular thickness between successive visits that exceed 6 m in pre-presbyopic individuals are therefore likely to reflect actual structural change. OCT measures are more variable in older individuals and it is advisable to take a series of scans so that outliers can be more easily identified.

Le trou maculaire (TM) est un défaut dans la rétine au niveau de la zone centrale de la macula (fovéa). Typiquement, les patients atteints de cette pathologie auront une diminution de leur acuité visuelle (AV) et de la métamorphopsie. Le traitement pour ces patients consistera en une vitrectomie par la pars plana (VPP) dans laquelle le vitré est retiré et remplacé par une tamponnade (SF6, C3F8, air ou huile de silicone). Après cette chirurgie, le succès chirurgical, soit de fermeture anatomique du TM, est très élevé. Toutefois, plus récemment, on s'aperçoit que malgré un taux de fermeture anatomique élevé, les résultats visuels après l'intervention chirurgicale ne sont pas aussi importants, d'où la nécessité d'identifier les facteurs pronostiques liés à la VPP dans les TM qui ont fermé chirurgicalement et de tenter de prédire les résultats visuels suivant la VPP. En ce qui concerne les TM qui n'ont pas fermé lors d'une première VPP, il est intéressant d'évaluer les résultats fonctionnels et anatomiques à la suite d'une VPP de reprise. Nos travaux ont démontré que les yeux avec une durée du TM plus courte, une taille du TM plus petite et une AV préopératoire plus élevée ont obtenu de meilleurs résultats visuels après une première chirurgie réussie. Nous avons par la suite produit un modèle hybride basé sur des données cliniques et des tomographies par cohérence optique haute définition (HD-OCT) préopératoires de TM, et ce, à l'aide de l'intelligence artificielle, afin de tenter de mieux prédire les résultats visuels. Les modèles basés sur les données cliniques ou les HD-OCT ont obtenu de bonnes performances discriminantes individuellement. Toutefois, la combinaison des deux modèles dans un modèle hybride n'a pas significativement amélioré les performances. Enfin, les TM qui subissaient une VPP de révision (non-fermeture chirurgicale à la première VPP) montraient une fermeture anatomique dans 85% des cas et avaient une amélioration de l'AV au fil du temps. / Idiopathic full thickness macular hole (MH) is a defect of all layers of the fovea. Typically, patients with this condition will experiment decreased visual acuity (VA) and metamorphopsia. Treatment for these patients will consist in a pars plana vitrectomy in which the vitreous is removed and replaced by a tamponade (SF6, C3F8, air or silicone oil). After this surgery, the surgical success, or anatomical closure of the MH, is very high. However, more recently, it has been noticed that despite a high anatomical closure rate, the visual results after the surgery are not quite as important, hence the need to identify the prognostic factors related to vitrectomy in patients with closed MH and to predict visual results following vitrectomy. For MH that did not close during the primary vitrectomy, it is interesting to assess the functional and anatomical results following a revision vitrectomy. Our works provided that eyes with shorter MH duration, smaller MH size and higher preoperative VA achieved better visual outcomes after successful MH surgery. We then produced a hybrid model based on clinical data and preoperative MH high-definition optical coherence tomography (HD-OCT), using artificial intelligence to try to better predict visual results. Both the clinical data and HD-OCT models had good discriminative performances. Combining both into a hybrid model did not significantly improve performance. Finally, MH that underwent revision vitrectomy (nonsurgical closure at primary vitrectomy) showed anatomical closure in 85% of cases and had VA improvement over the time.

No description available.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-09-26T15:36:47Z No. of bitstreams: 2 Dissertação - Letícia Pinto Coelho Jorge - 2017.pdf: 7825738 bytes, checksum: 4eca5b3a59512770f7384ddd353ddb59 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-27T11:21:13Z (GMT) No. of bitstreams: 2 Dissertação - Letícia Pinto Coelho Jorge - 2017.pdf: 7825738 bytes, checksum: 4eca5b3a59512770f7384ddd353ddb59 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-27T11:21:13Z (GMT). No. of bitstreams: 2 Dissertação - Letícia Pinto Coelho Jorge - 2017.pdf: 7825738 bytes, checksum: 4eca5b3a59512770f7384ddd353ddb59 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-09-19 / Macula lutea is the region of the retina where the yellow pigments lutein and zeaxanthin are concentrated. Oxidative damage seems to be an important factor for exacerbation of several retinal diseases, such as age-related macular degeneration, and a protective role of macular pigment has been postulated. The quantitative study of macular pigment and its distribution are possible through the determination of macular pigment optical density (MPOD). The objective of this study was to determine the mean MPOD value in a sample of the Brazilian population and to evaluate the influence of sex, age, ethnicity, smoking history and refractive status on MPOD values in this sample. A cross-sectional study was performed. Forty-two healthy patients had both eyes photographed using Visucam 500 (Carl Zeiss Meditec, Jena, Germany) in combination with the MPOD module. Four variables were obtained: maximum MPOD, mean MPOD, MPOD volume and MPOD area. Demographic data and life habits were collected. The mean MPOD value in the studied population was 0.13 density unit ± 0.02. MPOD values were not influenced by gender, smoking history or refraction. MPOD values were significantly higher among black patients when compared to caucasians. There was a positive but low correlation between mean MPOD and age. / A mácula lútea é a região da retina onde se concentram os pigmentos amarelos luteína e zeaxantina. Acredita-se que eles sejam um fator de proteção para doenças atribuídas ao estresse oxidativo, como degeneração macular relacionada à idade. O estudo quantitativo do pigmento macular e sua distribuição são possíveis por meio da medida da densidade óptica de pigmento macular (MPOD). O objetivo deste trabalho foi determinar o valor médio de MPOD em uma amostra da população brasileira e avaliar a influência dos fatores sexo, idade, etnia, histórico de tabagismo e status refracional nos valores de MPOD nesta amostra. Foi realizado estudo tipo corte transversal. Quarenta e dois pacientes saudáveis tiveram ambos os olhos fotografados utilizando o Visucam 500 (Carl Zeiss Meditec, Jena, Alemanha) em combinação com o módulo MPOD. Quatro variáveis foram obtidas: MPOD máxima, MPOD média, volume de MPOD e área de MPOD. Foram colhidos dados demográficos e hábitos de vida. O valor médio de MPOD nesta amostra da população brasileira foi de 0,13 unidade de densidade ± 0,02. Os valores de MPOD não foram influenciados pelo sexo, histórico de tabagismo ou refração. Os valores de MPOD foram significativamente maiores entre os pacientes negros, quando comparados aos brancos. Encontrou-se uma correlação positiva, porém baixa, entre o valor de MPOD médio e a idade.

However, this is by no means the limit. More knowledge on macular hole is needed. Continuous research in Hong Kong and other scientific communities are indispensable. A multicentre, multi-surgeon study should be contemplated and carried out prior to the generalization or application of this technique to all patients. / Over the last few years, we have collected essential clinical and laboratory data on indocyanine green (ICG) assisted internal limiting membrane (ILM) peeling in macular hole surgery in Hong Kong. With the intraoperative use of a small volume and low concentration of ICG, we have shown the usefulness and safety of this technique in improving the surgical outcomes, both antomically and functionally, in patients with macular hole of idiopathic type or in severely myopic eyes. Our randomized controlled trial demonstrated higher anatomical closure rate and better visual outcome with ICG assisted ILM peeling compared with no ILM peeling in primary idiopathic macular hole surgery. Our case-control study also demonstrated that ICG assisted ILM removal in macular hole surgery of severely myopic eyes gives promising anatomical closure rate and visual outcome comparable to emmetropic ones. / Kwok Kwan-ho. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 201-214). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Bei der Altersabhängigen Makuladegeneration (AMD) handelt es sich um eine Erkrankung des Auges, die die Macula lutea, die Stelle des schärfsten Sehens betrifft. Sie ist verbunden mit der Degeneration von RPE-Zellen, die zur Dystrophie von Photorezeptoren und damit zum Verlust des zentralen Sehvermögens führt. Eine ähnliche Pathophysiologie ist bei der sogenannten Retinalen Pigmentepitheldystrophie (RPED) des Hundes zu beobachten. Die Transplantation von gesunden RPE-Zellen in das betroffene Gebiet stellt eine vielversprechende Therapiemöglichkeit dar. Die Transplantatabstoßung als Kom-plikation schränkt die klinische Anwendung ein. Eine beim Patienten nach Transplantation lebenslang durchgeführte systemische Immunsuppression ist mit erheblichen Nebenwirkungen verbunden. Deshalb bietet die Gentherapie unter Einbezug immunsuppressiver Zytokine wie beispielsweise des Interleukin-10 (IL-10) eine Lösung. In der vorliegenden Arbeit wurde ein selbst konstruierter IL-10-Expressionsvektor (Plasmid pCIneoIL-10) mittels Gentransfer in humane RPE-Zellen in vitro eingebracht. Untersucht wurde die Wirkung des sezernierten IL-10 auf die Proliferation von allogenen T-Lymphozyten mit und ohne allogene Makrophagen als professionelle antigenpräsentierende Zellen (APC). Neben humanen Spender RPE-Zellen (Spender-hRPE-Zellen) wurde eine immortalisierte Permanent-Zelllinie (hTERT-RPE1-Zellen) eingesetzt, deren Hauptvorteil in einer gleichbleibend hohen Wachstumsrate lag. Als transientes Transfektions-system für den Transfer von IL-10-DNA in hRPE-Zellen wurden kationische Lipide gewählt. Drei verschiedene Lipidformulierungen wurden miteinander verglichen und das optimale Transfektionsreagenz:DNA-Verhältnis, mit dem die höchste Transfektionseffizienz erreicht werden konnte, evaluiert. Eine Transfektionseffizienz von 23,3 ± 9,0 % (hTERT-RPE1-Zellen) beziehungsweise 10,3 ± 4,5 % (Spender-hRPE-Zellen) konnte erreicht werden. Die Transfektion hatte weder einen negativen Einfluss auf die Vitalität der hRPE-Zellen, noch wurde der natürliche Zelltod, die Apoptose, erhöht. Die IL-10-mRNA-Expression wurde mittels RT-PCR nachgewiesen. Lediglich bei den transfizierten hRPE-Zellen konnte IL-10-mRNA gefunden werden. Mittels ELISA konnte das IL-10-Protein gemessen werden. Die Sekretion des IL-10 in den Kulturüberstand von transfizierten hRPE-Zellen wurde dafür über einen Zeitraum von 7 Tagen untersucht. Es konnte festgestellt werden, dass die maximale IL-10-Proteinkonzentration bei beiden Zelllinien am Tag 3 mit Werten von 10,3 ± 0,8 ng/ml (hTERT-RPE1-Zellen) und 3,1 ng/ml (Spender-hRPE-Zellen) lag. Es bestand überdies eine positive Korrelation zwischen Transfektionseffizienz und synthetisiertem IL-10. Es wurde außerdem gezeigt, dass durch Stimulation mit dem immunmodulatorischen Zytokin Interferon-gamma (IFN-g) hRPE-Zellen MHC Klasse II-Moleküle vermehrt exprimierten. Damit sind sie ebenso wie die Makrophagen zur Antigenpräsentation fähig. Die Wirkung des von den transfizierten hRPE-Zellen sezernierten IL-10 auf die Proliferation von T-Lymphozyten wurde zwischen Tag 2 und Tag 6 (hTERT-RPE1-Zellen) beziehungsweise zwischen Tag 2 und Tag 4 (Spender-hRPE-Zellen) photometrisch untersucht. Die Proliferation allogener T-Lymphozyten mit beziehungsweise ohne Makrophagen konnte durch das sezernierte IL-10 supprimiert werden. Bei den hTERT-RPE1-Zellen lag ohne die Anwesenheit von professionellen APC am Tag 6 eine signifikante Reduktion der T-Lymphozytenproliferation vor, während bei Kokultivierung mit Makrophagen Signifikanzen am Tag 5 und Tag 6 erkennbar waren. Die immunsuppressive Wirkung von IL-10 konnte mittels Anti-IL-10-Antikörper neutralisiert werden. Damit wurde bewiesen, dass die proliferations-supprimierende Wirkung auf IL-10 zurückzuführen war. Diese Ergebnisse könnten demnach neue Möglichkeiten zur Verhinderung einer Abstoßungsreaktion nach RPE-Zelltransplantation bei Patienten mit AMD eröffnen / Age-related macular degeneration (AMD) is a disease of eyes affecting the macula lutea, the area of the retina with the highest density of retinal pigment epithelial cells (RPE cells). The disease is characterized by degeneration of RPE cells resulting in dystrophy of photoreceptors and finally loss of central vision. Transplantation of healthy RPE cells is a promising possibility for therapy but rejection of the allotransplant limits clinical application. One way to avoid this complications is a systemic immunosuppression of the recipient but this is combined with many side effects. In this thesis a self-constructed IL-10 expression vector (plasmid pCIneoIL-10) has been transferred into human RPE cells in vitro by gene transfer. In addition to human donor RPE cells a permanent RPE cell line (hTERT-RPE1 cells) was employed. Kationic lipids were used as transient transfection system for transfer of pCIneoIL-10 into hRPE cells. Three different lipid formulations and various ratios of transfection reagent:DNA were evaluated for highest transfection efficacy. With the optimized protocols a transfection efficacy of 23,3 ± 9,0 % (hTERT-RPE1 cells) and 10,3 ± 4,5 % (donor hRPE cells) was achieved. A negative influence on the viability of the hRPE cells after transfection was not observed. The IL-10 mRNA expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR). Only in transfected hRPE cells the IL-10 mRNA-amplicon with 383 bp in size was found. Secretion of IL-10 protein in the cell culture supernatants of transfected hRPE cells was investigated using an enzyme-linked immunosorbent assay (ELISA) daily for 7 days. The IL-10 protein concentrations peaked at day 3 with 10,3 ± 0,8 ng/ml (hTERT-RPE1 cells) and 3,1 ng/ml (donor hRPE cells). The amount of secreted IL-10 positively correlated with transfection efficacy. After stimulation with the immunmodulatory cytokine interferon-gamma (IFN-g) the expression of MHC class II molecules on hRPE cells is increasing. Therefore they are able to present antigens similar to macrophages. Hence, the effects of recombinantly expressed IL-10 on the proliferation of allogeneic T lymphocytes were investigated both with and without allogeneic macrophages as professional antigen presenting cells (APC). Proliferation of T lymphocytes has been investigated colorimetrically between day 2 and day 6 (hTERT-RPE1 cells) and day 2 and day 4 (donor hRPE cells) respectively. The proliferation of allogeneic T lymphocytes with and without macrophages could be suppressed by the secreted IL-10. Signifikant reduction of proliferation was observed at day 6 in absence of professional APC (14,1 ± 1,1 % to 100% of untransfected control) and between day 5 (44,1 ± 4,9 %) and day 6 (37,4 ± 6,3%) in the presence of macrophages. It was possible to neutralize the immunosuppressive effect of IL-10 with anti-IL-10 antibodies. Proving that the suppressive effect of T lymphocyte proliferation was caused by IL-10. Thus, the specific IL-10 gene transfer into hRPE cells prior to transplantation may prevent rejection process and could prove a reliable method to help prevent loss of central vision due to AMD.

O comprometimento macular na retinopatia diabética pode ocorrer devido a isquemia macular, hemorragias maculares intra-retinianas e pré-retinianas, descolamento macular tracional, neovascularização e mais freqüentemente o edema macular. Cerca de 10% da população total de pacientes com diabete melito (DM) e 25% daqueles com mais de 20 anos de evolução da doença apresentam edema macular, constituindo a principal causa de perda visual neste grupo de pacientes(1-3). Em pacientes com DM tipo 1, o edema macular é visto raramente antes dos primeiros nove anos após o diagnóstico de DM(4), ao contrário de pacientes diabéticos tipo 2 cujo início da maculopatia é mais precoce. A detecção e o tratamento precoce do edema macular podem resultar na melhora ou na estabilização da visão. Novas teorias sobre a fisiopatologia do edema macular surgiram nos últimos anos e novas terapias como a triancinolona sub-tenoneana ou intravítrea, os anti-VEGFs e implantes intravítreos de liberação lenta de dexametasona foram testadas a partir destes conhecimentos. Este manuscrito propõe a revisão destes conceitos e em especial, dos novos aspectos terapêuticos. / The macular commitment on diabetic retinopathy can occur due to macular ischaemia, intraretinal or preretinal haemorrhages, tractional detachment, neovascularization and more frequently, macular edema. About 10% of the diabetic population and 25% of those with more than 20 years of illness, present macular edema, constituting the main cause of visual loss in this group of patients (1-3). In diabetic type 1 patients, macular edema is rarely seen before 9 years after the diagnostic(4). On the other hand, in diabetic type 2 patients, the maculopathy appears earlier. The detection and the precocious treatment of macular edema can result a visual improvement or stabilization. New theories about the fisiopathology of macular edema had appeared in the last years and new therapies such as sub-tenonean or intravitreal triamcinolone, the anti- VEGFs and slow liberation intravitreal implants of dexametazone were tested since this knowledge. This article considers the revision of these concepts and specially, new therapeutic aspects.