Estima-se que, em 2013, cerca de 382 milhões de pessoas eram portadoras de diabetes mundialmente. Já o diabetes mellitus do tipo 1 (DMT1) representa de 5-10% desse total de casos, cujo tratamento atual se pauta na administração de insulina exógena. Contudo, desde a publicação do protocolo de Edmonton, o transplante de ilhotas pancreáticas se apresenta como nova técnica no tratamento para o DMT1, inclusive obtendo a independência de insulina em alguns casos. Apesar disso, a escolha do sítio receptor ainda é essencial para diminuir efeitos adversos e permitir o acompanhamento do enxerto. Nesse sentido, destaca-se o transplante de ilhotas para a câmara anterior do olho, pois permite, além do restabelecimento do controle glicêmico, o estudo da fisiologia dos enxertos in vivo. Dessa forma, o objetivo foi estabelecer metodologia de isolamento e transplante de ilhotas de alta reprodutibilidade e baixo custo, utilizando a câmara anterior do olho como sítio receptor. O isolamento foi realizado via injeção de solução de colagenase (1 mg/mL via ducto colédoco) em camundongos machos C57BL/6 hígidos de 8 semanas de idade e posterior transplante dessas ilhotas para camundongos machos da mesma espécie com diabetes induzido por injeção de aloxana (60 mg/kg, i.v.). Esses camundongos foram submetidos a infusão de aproximadamente 250 equivalentes de ilhotas (IEQs) para a câmara anterior do olho e tiveram sua glicemia e alteração de massa corpórea acompanhadas por 14 dias após o transplante. Também foi realizado teste de tolerância a glicose via injeção de solução de glicose (2g/kg i.p.) e realização da curva glicêmica. Obteve-se, na etapa de padronização, que a adição de 0,5% (%p/v) de albumina de soro bovino à solução de colagenase foi capaz de aumentar o número de IEQs isolados por animal. Quanto ao transplante, obteve-se que 50% dos animais submetidos à técnica tiveram diminuição significativa na sua glicemia (172,5 ± 6,4 mg/dL), quando comparados com o grupo controle diabético (582,8 ± 27,5 mg/dL) (p < 0,05). Entretanto, todos os animais tiveram aumento significativo da massa corpórea no período de acompanhamento e glicemia de jejum significativamente menor que os animais diabéticos (p < 0,05). Ademais, a curva glicêmica dos animais que tiveram transplante considerado bem sucedido, no teste de tolerância a glicose, se aproxima da curva do grupo controle sadio. Conclui-se que o modelo de transplante de ilhotas pancreáticas para a câmara anterior do olho foi bem estabelecido neste projeto, confirmado pelos resultados que evidenciam o transplante de ilhotas funcionais capazes de reduzir sensivelmente a glicemia e promover o ganho de peso em camundongos diabéticos. / It is estimated that, in 2013, around 382 million people had diabetes worldwide. Of that number, 5-10% represented cases of T1DM, which treatment is based in the administration of exogenous insulin. However, since the Edmonton protocol was published, islet transplantation presented itself as novel technique for T1DM treatment, achieving insulin independence in some cases. Although, recipient site choice is still essential to diminish side effects and enable graft follow up. In that sense, transplantation to the anterior chamber of the eye stands out, since it allows, beyond the reestablishment of glycemic control, study of islet physiology in vivo. That way, the objective was to establish a low cost and high reproducible model of islet isolation and transplantation, using the anterior chamber of the eye as receptor site. Islet isolation was made by injection of collagenase solution (1 mg/mL via common bile duct) in 8 week old healthy male C57BL/6 mice and followed by transplantation of these islets to male mice of the same age and species with diabetes induced by alloxan injection (60 mg/kg i.v.). These mice were subject of 250 islet equivalents (IEQs) infusion to the anterior chamber of the eye and had their blood glucose and change in body mass monitored for 14 days after transplantation. A glucose tolerance test (GTT) was also made, by injection of glucose solution (2g/kg i.p.) and a glycemic curve was plotted. In the standardization period, was observed that the addition of 0,5% (%w/v) bovine serum albumin is capable of increasing the number of IEQs isolated from each animal. About the transplants, was obtained that 50% of animals subject to transplantation had their blood glucose decreased significantly (172,5 ± 6,4 mg/dL), when compared to the diabetic control group (582,8 ± 27,5 mg/dL) (p < 0,05). However, all animals subject to the procedure had significant body mass increase, when compared to the same control group and fasting blood glucose significantly lower than diabetic animals (p < 0,05). Moreover, the glycemic curve of animals, who had their transplantation considered successful, was similar to that found in healthy control animals, in the GTT. We conclude that the model of transplant to the anterior chamber of the eye is well established in this project, which is confirmed by results that shows transplantation of functional islets, capable of promoting a significant decrease in blood glucose and an increase in total body mass in diabetic animals.

Depuis plus d’un siècle, l’œil est décrit comme un site immuno-privilégié : les réponses immunitaires sont fortement contrôlées localement et un mécanisme de déviation immunitaire peut être induit en périphérie après injection d’antigènes en chambre antérieure (ACAID, Anterior Chamber-Associated Immune Deviation). Mieux caractériser le statut immunitaire de l’œil, et en particulier de la rétine, est devenu un enjeu majeur à l’heure actuel au vu des nombreuses approches thérapeutiques innovantes pour le traitement de pathologies rétiniennes. L’objectif de mon travail de thèse a été d’étudier l’impact immunitaire périphérique d’une injection sous-rétinienne, tout d’abord dans un contexte antigénique simple, puis dans le cadre d’un transfert de gène par virus adéno-associé recombinant. En utilisant un modèle murin, j’ai pu démontrer que l’injection sous-rétinienne de peptides induit une inhibition des réponses lymphocytaires Th1/Tc1, Th2/Tc2, Th17/Tc17 en périphérie, de manière similaire à l’ACAID. Néanmoins, ce privilège immunitaire de la zone sous-rétinienne présente ses limites puisque l’injection sous-rétinienne de fortes doses d’AAV8 induit une réponse anti-transgène pro-inflammatoire en périphérie, accompagnée d’une altération locale de la structure rétinienne. Ce travail de thèse ouvre par ailleurs des voies de recherche quant à des approches d’immuno-modulation spécifiques. A terme, ces résultats devraient permettre d’optimiser l’immuno-monitorage des patients dans les essais cliniques. / For over a century, the eye has been described as an immune privileged site : immune responses are tighly controlled locally and an immune deviation mechanism is induced in periphery after the injtection of antigens in the anterior chamber (ACAID, Anterior Chamber-Associated Immune Deviation). A better comprehension of the immune status of the eye, especially the retina, has become a major concern regarding emerging and innovative therapeutic approaches to treat retinal pathologies. The aim of my work was to study the peripheral immune consequences of a subretinal injection, first in an simple antigenic context, and second in the context of adeno-associated virus-mediated gene transfer. In a murine model, I have shown that the subretinal injection of peptides induces an inhbition of Th1/Tc1, Th2/Tc2, Th17/Tc17 immune responses, similarly to ACAID. However, such an immune privilege has its limits. Indeed, the subretinal injection of high doses of AAV8 induces a pro-inflammatory anti-transgene immune response, along with a local alteration of the retinal structure. This work also provides new leads for specific approaches of immune modulation. On the long term, these results should provide new insights in optimizing the immuno-monitoring of patients enrolled in clinical trials.

<p>Katarakta i glaukom su po svom toku hronične i progresivne bolesti koji predstavljaju dva vodeća uzroka slepila u svetu. Obe bolesti su karakteristične za stariju životnu dob i često se sreću zajedno kod iste osobe. Katarakta podrazumeva hirur&scaron;ko lečenja, dok se lečenje glaukoma zasniva na snižavanju visine intraokularnog pritiska (IOP), medikamentoznim i hirur&scaron;kim sredstvima. Cilj ovog istraživanja bio je da se utvrdi da li i u kojoj meri dolazi promene IOP-a i dubine prednje komore oka (ACD) nakon operacije katarakte, da li su preoperativne vrednosti IOP-a i ACD povezane sa postoperativnom visinom IOP-a i da li postoje razlike u ovim pojavama kod osoba operisanih od katarakte sa i bez prisutnog primarnog glaukoma otvorenog ugla (POAG). Rezultati ove studije pokazuju da postoji statistički značajno sniženje IOP-a 6 meseci nakon operacije katarakte. U poređenju sa preoperativnim vrednostima, ono iznosi prosečno 1,2 mmHg (7,5%) u grupi pacijenata bez POAG i 1,24 mmHg (6,49%) u grupi pacijenata sa POAG. Između dve grupe pacijenata nije postojala razlika u stepenu sniženja (p&gt;0,05). Ustanovljen je statistički značajano veći (p&lt;0,05) porast dubine ACD u grupi pacijenata sa POAG (1,03 mm; 34,8%) u odnosu pacijente bez glaukomske bolesti (0,92 mm; 30,37%). Rezultati korelacione analize, kao i regresionih univarijantih i multivarijantnih modela, pokazali su da statistički značajna povezanost postoji između preoperativne visine IOP-a i njegovog sniženja nakon operacije katarakte. Povezanost je bila pozitivnog smera i nije se uočena značajna razlika između obe grupe pacijenata. Ustanovljena je i pozitiva korelacija, bez statistički značajne razlike u obe grupe pacijenata, između PD indeksa (odnos preoperativnog IOP-a i ACD) i promene IOP-a nakon operacije katarakte. Preoperativna dubina prednje komore oka nije ispoljila povezanost sa postoperativnom promenom IOP-a. Na&scaron;a studija je pokazala statistički značajno postoperativno sniženje IOP-a i povećanje dubine ACD nakon operacije katarakte, koja se održava &scaron;est meseci nakon operacije katarakte. Nije ustanovljena razlika u redukciji IOP-a između pacijenata sa i bez POAG. Ustanovljena je pozitivna korelacija preoperativne visine IOP-a i PD indeksa sa postoperativnom promenom IOP-a, &scaron;to može biti od koristi prilikom odluke o optimalnom lečenju katarakte kod pacijenata sa POAG.</p> / <p>Cataract and glaucoma are chronic and progressive diseases and they are two of the leading causes of blindness wold wide. Both diseases are typical for an older age and often coincide within the same person. Treatment of cataract is surgical, while glaucoma treatment is based on lowering the level of intraocular pressure (IOP) with various medical and surgical options. The aims of this research were to determine whether there is a change in IOP and in the depth of anterior chamber of the eye (ACD) after cataract surgery, whether preoperative values of IOP and ACD are related to postoperative IOP values and to determine if there are differences in these events between people operated from cataract with or without primary open angle glaucoma (POAG). Results of this study show that there is a statistically significant decrease of IOP six months after cataract surgery. Comparing with preoperative values, it was found to be 1.2 mmHg (7.5%) in group of patients without POAG, and 1.24 mmHg (6.49%) in patients with POAG. There was no statistically significant difference found between two groups of patients (p&lt;0.05). Group of patients with POAG had significantly higher (p&lt;0.05) deepening of ACD (1.03 mm; 34.8%), in comparison with patients with no glaucoma (0.92 mm; 30.37%). Results of correlation analysis, as well as univariate and multivariate regression models, have shown significant correlation of preoperative IOP and its reduction after cataract surgery. Correlation was of positive direction with no statistically significant differences among two groups of patients. There was a positive correlation found, with no statistically significant differences in both groups of patients, between PD index (ratio of preoperative IOP and ACD) and IOP change after cataract surgery. Preoperative anterior chamber depth has shown no correlation with postoperativeIOP change. Our study showed statisticallysignificant postoperative reduction ofIOP and deepening of ACD, lasting for six months aftercataract surgery. No difference inpostoperativeIOP decrease has been detected betweenpatients with or without POAG. A positivecorrelation of preoperativeIOP height and PDindex with postoperativeIOP change has beenestablished, which mayprove usefulfordecision of optimal treatmentof cataract among POAG patients.</p>

OBJECTIVE: Ocular function is fundamental for environmental adaptation and survival capacity. Growth factors are necessary for a mature eyeball, needed for adequate vision. However, the consequences of the deficiency of circulating growth hormone (GH) and its effector insulin-like growth factor I (IGF-I) on the physical aspects of the human eye are still debated. A model of untreated isolated GH deficiency (IGHD), with low but measurable serum GH, may clarify this issue. The aim of this study was to assess the ocular aspects of adult IGHD individuals who have never received GH therapy. DESIGN: Cross sectional study. METHODS: Setting University Hospital, Federal University of Sergipe, Brazil. Patients: Twenty-five adult (13 males, mean age 50.1 years, range 26 to 70 years old) IGHD subjects homozygous for a null mutation (c.57+1G>A) in the GHRH receptor gene, and 28 (15 males, mean age 51.1 years, range 26 to 67 years old) controls were submitted to an endocrine and ophthalmological assessment. Forty-six IGHD and 50 control eyes were studied. Main outcome measures: Visual acuity, intraocular pressure (IOP), refraction (spherical equivalent), ocular axial length (AL), anterior chamber depth (ACD),lens thickness (LT), vitreous depth (VD), mean corneal curvature (CC) and central corneal thickness (CCT). RESULTS: IGHD subjects exhibited unmeasurable serum IGF-I levels, similar visual acuity, intraocular pressure and LT, higher values of spherical equivalent and CC, and lower measures of AL, ACD, VD and CCT in comparison to controls, but within their respective normal ranges. While mean stature in IGHD group was 78 % of the control group, mean head circumference was 92 % and axial AL was 96 %. CONCLUSIONS: These observations suggest mild ocular effects in adult subjects with severe IGF-I deficiency due to non-treated IGHD. / OBJETIVO: A função ocular é fundamental para a adaptação ambiental e a capacidade de sobrevivência. Fatores de crescimento são julgados necessáriospara alcançar um globo ocular maduro, e conseqüente visão adequada. No entanto, as consequências da deficiência isoladadohormônio de crescimento circulante (GH) edo seu efetor, o fator de crescimento semelhante à insulina I (IGF-I) nos aspectos físicos do olho humano ainda são debatidas. Um modelo de deficiência isolada de GH não tratada (DIGH) pode esclarecer esta questão. O objetivo deste estudo foi avaliar os aspectos físicos do globo ocular de indivíduos adultos com DIGH que nunca receberam terapia com GH. DESENHO: Estudo transversal. MÉTODOS: Ambiente: Hospital Universitário, Universidade Federal de Sergipe, Brasil. Pacientes: 25 indivíduosadultos (13 homens,com média de idade de 50,1 anos, entre 26 e 70 anos), com DIGH homozigotos para uma mutação nula (c.57 + 1G> A) no gene do receptorGHRH do grupo DIGH e 28 controles (15 homens, com média de idade de 51,1 anos, entre 26 e 67 anos), pareados, foram submetidos à avaliação endócrina e oftalmológica. Principais medidas: acuidade visual(AV), pressão intraocular(PIO),refração (equivalente esférico, EE), comprimento axial ocular (CA), profundidade da câmara anterior(PCA), medida da espessura do cristalino(EC), profundidade do vítreo(PV), curvatura corneana média(CCM) e espessura central corneana(ECC). RESULTADOS:Indivíduos com DIGH apresentaram IGF-I sérico não mensurável, similarAV, PIO e EC, valores mais altos doEEe CCM, e menores valores do CA, PCA, PV e ECC em comparação com os controles, mas dentro das respectivas faixas normais. Enquanto a estaturamédia no grupo DIGH foi de 78% do grupo de controle, a média da circunferência da cabeça foi de 92% e a média docomprimento axial foi de 96%. CONCLUSÃO: Essas observações sugerem efeitos oculares discretosem indivíduos adultos com grave deficiência de IGF-I devido à DIGH não tratada.

OBJECTIVE: Ocular function is fundamental for environmental adaptation and survival capacity. Growth factors are necessary for a mature eyeball, needed for adequate vision. However, the consequences of the deficiency of circulating growth hormone (GH) and its effector insulin-like growth factor I (IGF-I) on the physical aspects of the human eye are still debated. A model of untreated isolated GH deficiency (IGHD), with low but measurable serum GH, may clarify this issue. The aim of this study was to assess the ocular aspects of adult IGHD individuals who have never received GH therapy. DESIGN: Cross sectional study. METHODS: Setting University Hospital, Federal University of Sergipe, Brazil. Patients: Twenty-five adult (13 males, mean age 50.1 years, range 26 to 70 years old) IGHD subjects homozygous for a null mutation (c.57+1G>A) in the GHRH receptor gene, and 28 (15 males, mean age 51.1 years, range 26 to 67 years old) controls were submitted to an endocrine and ophthalmological assessment. Forty-six IGHD and 50 control eyes were studied. Main outcome measures: Visual acuity, intraocular pressure (IOP), refraction (spherical equivalent), ocular axial length (AL), anterior chamber depth (ACD),lens thickness (LT), vitreous depth (VD), mean corneal curvature (CC) and central corneal thickness (CCT). RESULTS: IGHD subjects exhibited unmeasurable serum IGF-I levels, similar visual acuity, intraocular pressure and LT, higher values of spherical equivalent and CC, and lower measures of AL, ACD, VD and CCT in comparison to controls, but within their respective normal ranges. While mean stature in IGHD group was 78 % of the control group, mean head circumference was 92 % and axial AL was 96 %. CONCLUSIONS: These observations suggest mild ocular effects in adult subjects with severe IGF-I deficiency due to non-treated IGHD. / OBJETIVO: A função ocular é fundamental para a adaptação ambiental e a capacidade de sobrevivência. Fatores de crescimento são julgados necessáriospara alcançar um globo ocular maduro, e conseqüente visão adequada. No entanto, as consequências da deficiência isoladadohormônio de crescimento circulante (GH) edo seu efetor, o fator de crescimento semelhante à insulina I (IGF-I) nos aspectos físicos do olho humano ainda são debatidas. Um modelo de deficiência isolada de GH não tratada (DIGH) pode esclarecer esta questão. O objetivo deste estudo foi avaliar os aspectos físicos do globo ocular de indivíduos adultos com DIGH que nunca receberam terapia com GH. DESENHO: Estudo transversal. MÉTODOS: Ambiente: Hospital Universitário, Universidade Federal de Sergipe, Brasil. Pacientes: 25 indivíduosadultos (13 homens,com média de idade de 50,1 anos, entre 26 e 70 anos), com DIGH homozigotos para uma mutação nula (c.57 + 1G> A) no gene do receptorGHRH do grupo DIGH e 28 controles (15 homens, com média de idade de 51,1 anos, entre 26 e 67 anos), pareados, foram submetidos à avaliação endócrina e oftalmológica. Principais medidas: acuidade visual(AV), pressão intraocular(PIO),refração (equivalente esférico, EE), comprimento axial ocular (CA), profundidade da câmara anterior(PCA), medida da espessura do cristalino(EC), profundidade do vítreo(PV), curvatura corneana média(CCM) e espessura central corneana(ECC). RESULTADOS:Indivíduos com DIGH apresentaram IGF-I sérico não mensurável, similarAV, PIO e EC, valores mais altos doEEe CCM, e menores valores do CA, PCA, PV e ECC em comparação com os controles, mas dentro das respectivas faixas normais. Enquanto a estaturamédia no grupo DIGH foi de 78% do grupo de controle, a média da circunferência da cabeça foi de 92% e a média docomprimento axial foi de 96%. CONCLUSÃO: Essas observações sugerem efeitos oculares discretosem indivíduos adultos com grave deficiência de IGF-I devido à DIGH não tratada.