An Advocacy for the Use of 3D Stem Cell Culture Systems for the Development of Regenerative Medicine: An Emphasis on Photoreceptor Generation

The availability of stem cells is of great promise to study early developmental stages and to generate adequate cells for cell transfer therapies. Although many researchers using stem cells were successful in dissecting intrinsic and extrinsic mechanisms and in generating specific cell phenotypes, few of the stem cells or the differentiated cells show the capacity to repair a tissue. Advances in cell and stem cell cultivation during the last years made tremendous progress in the generation of bona fide differentiated cells able to integrate into a tissue after transplantation, opening new perspectives for developmental biology studies and for regenerative medicine. In this review, we focus on the main works attempting to create in vitro conditions mimicking the natural environment of CNS structures such as the neural tube and its development in different brain region areas including the optic cup. The use of protocols growing cells in 3D organoids is a key strategy to produce cells resembling endogenous ones. An emphasis on the generation of retina tissue and photoreceptor cells is provided to highlight the promising developments in this field. Other examples are presented and discussed, such as the formation of cortical tissue, the epithelial gut or the kidney organoids. The generation of differentiated tissues and well-defined cell phenotypes from embryonic stem (ES) cells or induced pluripotent cells (iPSCs) opens several new strategies in the field of biology and regenerative medicine. A 3D organ/tissue development in vitro derived from human cells brings a unique tool to study human cell biology and pathophysiology of an organ or a specific cell population. The perspective of tissue repair is discussed as well as the necessity of cell banking to accelerate the progress of this promising field.

Targeting iron-mediated retinal degeneration by local delivery of transferrin.

Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress.

CONVERSION TO AFLIBERCEPT THERAPY VERSUS CONTINUING WITH RANIBIZUMAB THERAPY FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION DEPENDENT ON MONTHLY RANIBIZUMAB TREATMENT.

PURPOSE: To compare the effects of converting to aflibercept therapy with continuing ranibizumab therapy in eyes with neovascular age-related macular degeneration requiring monthly ranibizumab treatment. METHODS: Patients were selected from the 104 patients (115 eyes) already enrolled in an "Observe and Plan" prospective case series that included treating neovascular age-related macular degeneration with ranibizumab for 24 months. Patients who still needed monthly retreatment at the end of a 2-year study were randomized to either continue ranibizumab therapy or to convert to aflibercept therapy. Outcome measures included average interval between treatments, resolution of exudative signs, number of retreatments, and change in visual acuity over 12 months (the third treatment year). RESULTS: Nineteen patients (21 eyes) met the inclusion criteria. Ten eyes were randomized to receive aflibercept, and 11 eyes remained on ranibizumab. Groups were balanced for baseline characteristics. Outcomes were similar in the 2 groups over a 12-month study duration, with no statistical difference. CONCLUSION: This comparative pilot study suggests that neovascular age-related macular degeneration requiring monthly retreatment with ranibizumab may respond in similar ways to both ranibizumab and aflibercept treatment. Larger sample sizes would be needed to confirm this observation.

Integrating quantitative proteomics and metabolomics in a cellular model of diabetic retinopathy

Diabetic retinopathy is the leading cause of visual loss in individuals under the age of 55. Most investigations into the pathogenesis of diabetic retinopathy have been concentrated on the neural retina since this is where clinical lesions are manifested. Recently, however, various abnormalities in the structural and secretory functions of retinal pigment epithelium that are essential for neuroretina survival, have been found in diabetic retinopathy. In this context, here we study the effect of hyperglycemic and hypoxic conditions on the metabolism of a human retinal pigment epithelial cell line (ARPE-19) by integrating quantitative proteomics using tandem mass tagging (TMT), untargeted metabolomics using MS and NMR, and 13C-glucose isotopic labeling for metabolic tracking. We observed a remarkable metabolic diversification under our simulated in vitro hyperglycemic conditions of diabetes, characterized increased flux through polyol pathways and inhibition of the Krebs cycle and oxidative phosphorylation. Importantly, under low oxygen supply RPE cells seem to consume rapidly glycogen storages and stimulate anaerobic glycolysis. Our results therefore pave the way to future scenarios involving new therapeutic strategies addressed to modulating RPE metabolic impairment, with the aim of regulating structural and secretory alterations of RPE. Finally, this study shows the importance of tackling biomedical problems by integrating metabolomic and proteomics results.

Functional impact of genetic variation on gene expression

Numerous links between genetic variants and phenotypes are known and genome-wide association studies dramatically increased the number of genetic variants associated with traits during the last decade. However, how changes in the DNA perturb the molecular mechanisms and impact on the phenotype of an organism remains elusive. Studies suggest that many traitassociated variants are in the non-coding region of the genome and probably act through regulation of gene expression. During my thesis I investigated how genetic variants affect gene expression through gene regulatory mechanisms. The first chapter was a collaborative project with a pharmaceutical company, where we investigated genome-wide copy number variation (CNVs) among Cynomolgus monkeys (Macaca fascicularis) used in pharmaceutical studies, and associated them to changes in gene expression. We found substantial copy number variation and identified CNVs linked to tissue-specific expression changes of proximal genes. The second and third chapters focus on genetic variation in humans and its effects on gene regulatory mechanisms and gene expression. The second chapter studies two human trios, where the allelic effects of genetic variation on genome-wide gene expression, protein-DNA binding and chromatin modifications were investigated. We found abundant allele specific activity across all measured molecular phenotypes and show extended coordinated behavior among them. In the third chapter, we investigated the impact of genetic variation on these phenotypes in 47 unrelated individuals. We found that chromatin phenotypes are organized into local variable modules, often linked to genetic variation and gene expression. Our results suggest that chromatin variation emerges as a result of perturbations of cis-regulatory elements by genetic variants, leading to gene expression changes. The work of this thesis provides novel insights into how genetic variation impacts gene expression by perturbing regulatory mechanisms. -- De nombreux liens entre variations génétiques et phénotypes sont connus. Les études d'association pangénomique ont considérablement permis d'augmenter le nombre de variations génétiques associées à des phénotypes au cours de la dernière décennie. Cependant, comprendre comment ces changements perturbent les mécanismes moléculaires et affectent le phénotype d'un organisme nous échappe encore. Des études suggèrent que de nombreuses variations, associées à des phénotypes, sont situées dans les régions non codantes du génome et sont susceptibles d'agir en modifiant la régulation d'expression des gènes. Au cours de ma thèse, j'ai étudié comment les variations génétiques affectent les niveaux d'expression des gènes en perturbant les mécanismes de régulation de leur expression. Le travail présenté dans le premier chapitre est un projet en collaboration avec une société pharmaceutique. Nous avons étudié les variations en nombre de copies (CNV) présentes chez le macaque crabier (Macaca fascicularis) qui est utilisé dans les études pharmaceutiques, et nous les avons associées avec des changements d'expression des gènes. Nous avons découvert qu'il existe une variabilité substantielle du nombre de copies et nous avons identifié des CNVs liées aux changements d'expression des gènes situés dans leur voisinage. Ces associations sont présentes ou absentes de manière spécifique dans certains tissus. Les deuxième et troisième chapitres se concentrent sur les variations génétiques dans les populations humaines et leurs effets sur les mécanismes de régulation des gènes et leur expression. Le premier se penche sur deux trios humains, père, mère, enfant, au sein duquel nous avons étudié les effets alléliques des variations génétiques sur l'expression des gènes, les liaisons protéine-ADN et les modifications de la chromatine. Nous avons découvert que l'activité spécifique des allèles est abondante abonde dans tous ces phénotypes moléculaires et nous avons démontré que ces derniers ont un comportement coordonné entre eux. Dans le second, nous avons examiné l'impact des variations génétiques de ces phénotypes moléculaires chez 47 individus, sans lien de parenté. Nous avons observé que les phénotypes de la chromatine sont organisés en modules locaux, qui sont liés aux variations génétiques et à l'expression des gènes. Nos résultats suggèrent que la variabilité de la chromatine est due à des variations génétiques qui perturbent des éléments cis-régulateurs, et peut conduire à des changements dans l'expression des gènes. Le travail présenté dans cette thèse fournit de nouvelles pistes pour comprendre l'impact des différentes variations génétiques sur l'expression des gènes à travers les mécanismes de régulation.

Modifier Genes as Therapeutics: The Nuclear Hormone Receptor Rev Erb Alpha (Nr1d1) Rescues Nr2e3 Associated Retinal Disease

Nuclear hormone receptors play a major role in many important biological processes. Most nuclear hormone receptors are ubiquitously expressed and regulate processes such as metabolism, circadian function, and development. They function in these processes to maintain homeostasis through modulation of transcriptional gene networks. In this study we evaluate the effectiveness of a nuclear hormone receptor gene to modulate retinal degeneration and restore the integrity of the retina. Currently, there are no effective treatment options for retinal degenerative diseases leading to progressive and irreversible blindness. In this study we demonstrate that the nuclear hormone receptor gene Nr1d1 (Rev-Erba) rescues Nr2e3- associated retinal degeneration in the rd7 mouse, which lacks a functional Nr2e3 gene. Mutations in human NR2E3 are associated with several retinal degenerations including enhanced S cone syndrome and retinitis pigmentosa. The rd7 mouse, lacking Nr2e3, exhibits an increase in S cones and slow, progressive retinal degeneration. A traditional genetic mapping approach previously identified candidate modifier loci. Here, we demonstrate that in vivo delivery of the candidate modifier gene, Nr1d1 rescues Nr2e3 associated retinal degeneration. We observed clinical, histological, functional, and molecular restoration of the rd7 retina. Furthermore, we demonstrate that the mechanism of rescue at the molecular and functional level is through the re-regulation of key genes within the Nr2e3-directed transcriptional network. Together, these findings reveal the potency of nuclear receptors as modulators of disease and specifically of NR1D1 as a novel therapeutic for retinal degenerations.

People favour imperfect catching by assuming a stable world

The visual angle that is projected by an object (e.g. a ball) on the retina depends on the object's size and distance. Without further information, however, the visual angle is ambiguous with respect to size and distance, because equal visual angles can be obtained from a big ball at a longer distance and a smaller one at a correspondingly shorter distance. Failure to recover the true 3D structure of the object (e.g. a ball's physical size) causing the ambiguous retinal image can lead to a timing error when catching the ball. Two opposing views are currently prevailing on how people resolve this ambiguity when estimating time to contact. One explanation challenges any inference about what causes the retinal image (i.e. the necessity to recover this 3D structure), and instead favors a direct analysis of optic flow. In contrast, the second view suggests that action timing could be rather based on obtaining an estimate of the 3D structure of the scene. With the latter, systematic errors will be predicted if our inference of the 3D structure fails to reveal the underlying cause of the retinal image. Here we show that hand closure in catching virtual balls is triggered by visual angle, using an assumption of a constant ball size. As a consequence of this assumption, hand closure starts when the ball is at similar distance across trials. From that distance on, the remaining arrival time, therefore, depends on ball's speed. In order to time the catch successfully, closing time was coupled with ball's speed during the motor phase. This strategy led to an increased precision in catching but at the cost of committing systematic errors.

Oeil et cancer

Résumé Le cancer implique rarement l'oeil et risque d'être reconnu tardivement. Les tumeurs intraoculaires primaires les plus fréquentes sont le rétinoblastome chez l'enfant et le mélanome uvéal chez l'adulte.Le diagnostic différentiel d'une baisse de vision dans un contexte de cancer systémique est varié. Des métastases uvéales sont souvent associées au cancer du sein ou du poumon. Un masquerade syndrome est l'atteinte oculaire, pseudo-inflammatoire, d'un lymphome primaire non hodgkinien du système nerveux central. Un traitement oncologique médicamenteux ou radique peut induire une toxicité, souvent rétinienne. Les syndromes paranéoplasiques, rares, sont causés par des anticorps anticancéreux réagissant contre la rétine. Si le cancer touche l'oeil, référer le patient rapidement vers un centre spécialisé pourra faire la différence aux niveaux pronostiques vital et visuel. Abstract Cancer involves so rarely the eye that it may be recognized late. The most frequent primary intra-ocular tumours are retinoblastoma in small children and uveal melanoma in adults.Vision loss in systemic cancer has a varied differential diagnosis. Uveal metastases are most often associated with breast cancer, but can herald lung carcinoma. Masquerade syndrome looks like infllammation but represents the ocular involvement of primary CNS non-Hodgkin lymphoma. Systemic cancer drugs, as well as radiotherapy, can cause ocular toxicity, mostly at the retina. In the rare paraneoplastic syndromes, patient's cancer antibodies cross-react with retinal antigens, leading to severe vision loss. When cancer involves the eye, a fast referral into specialized care can signifiicantly improve visual and vital prognosis.

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

Cancer therapy-related toxicity in the immature testis

Infertility is a common late effect of childhood cancer treatment. Testicular toxicity can clinically be first detected after the onset of pubertal maturation of the patients when the testis does not grow, spermatogenesis does not initiate and serum levels of gonadotrophins rise. Improved prognosis for childhood cancer has resulted in a growing number of childhood cancer survivors with late effects. In our study, we developed novel tools for detecting cancer therapy-related testicular toxicity during development. By using these methods the effects of the tyrosine kinase inhibitor imatinib mesylate, chemotherapy agent doxorubicin and irradiation on testicular development were investigated in rat and monkey. Patients with chronic myeloid leukemia and some patients with acute lymphoblastic leukemia have fusion gene BCR-ABL which codes for abnormal tyrosine kinase protein. Imatinib mesylate (Glivec®) inhibits activity of this protein. In addition, imatinib inhibits the action of the c-kit and PDGF –receptors, which are both important for the survival and proliferation of the spermatogonial stem cell pool. Imatinib exposure during prepubertal development disturbed the development and the growth of the testis. Spermatogonial stem cells were also sensitive to the toxic effects of doxorubicin and irradiation during the initiation phase of spermatogenesis. In addition, the effect of the treatment of acute lymphoblastic leukemia on germ cell numbers and recovery of reproductive functions after sexual maturation was investigated. Therapy for childhood acute lymphoblastic leukemia seldom results in infertility. The present study gives new information on the mechanisms by which cancer treatments exert their gonadal toxicity in immature testis.

Intravitreal docosahexaenoic acid in a rabbit model: preclinical safety assessment

Purpose The purpose of the present study was to evaluate the retinal toxicity of a single dose of intravitreal docosahexaenoic acid (DHA) in rabbit eyes over a short-term period. Methods Sixteen New Zealand albino rabbits were selected for this pre-clinical study. Six concentrations of DHA (Brudy Laboratories, Barcelona, Spain) were prepared: 10 mg/50 µl, 5 mg/50 µl, 2'5 mg/50 µl, 50 µg/50 µl, 25 µg/50 µl, and 5 µg/50 µl. Each concentration was injected intravitreally in the right eye of two rabbits. As a control, the vehicle solution was injected in one eye of four animals. Retinal safety was studied by slit-lamp examination, and electroretinography. All the rabbits were euthanized one week after the intravitreal injection of DHA and the eyeballs were processed to morphologic and morphometric histological examination by light microscopy. At the same time aqueous and vitreous humor samples were taken to quantify the concentration of omega-3 acids by gas chromatography. Statistical analysis was performed by SPSS 21.0. Results Slit-lamp examination revealed an important inflammatory reaction on the anterior chamber of the rabbits injected with the higher concentrations of DHA (10 mg/50 µl, 5 mg/50 µl, 2'5 mg/50 µ) Lower concentrations showed no inflammation. Electroretinography and histological studies showed no significant difference between control and DHA-injected groups except for the group injected with 50 µg/50 µl. Conclusions Our results indicate that administration of intravitreal DHA is safe in the albino rabbit model up to the maximum tolerated dose of 25 µg/50 µl. Further studies should be performed in order to evaluate the effect of intravitreal injection of DHA as a treatment, alone or in combination, of different retinal diseases.

Tumores colorretais hereditários

Cerca de 4% a 15% dos tumores colorretais são hereditários e divididos em dois grupos: polipose adenomatosa familiar (FAP) e câncer colorretal hereditário sem polipose (HNPCC). Ambas são doenças autossômicas dominantes, com transmissão vertical, geração após geração, sem preferência por sexo. A FAP tem penetrância praticamente completa, caracterizada por mais de cem pólipos adenomatosos no intestino grosso, que aparecem em geral após a puberdade e se transformam em câncer em todos os casos não tratados, levando o paciente ao óbito em tomo dos 45 anos de idade. Manifestações extracolônicas são comuns, tais como: pólipos em estômago e duodeno, sarcomas abdominais, pigmentação de retina, osteomas, entre outras. A FAP é causada por mutação no gene APC, que está localizado no cromossomo 5q. Seu tratamento é basicamente cirúrgico, com retirada do intestino grosso, podendo-se preservar o reto, se este não apresentar muitos pólipos. O HNPCC tem penetrância em torno de 80% e não apresenta os pólipos benignos como na FAP, que permitem identificar pacientes com o fenótipo da doença. Geralmente, o diagnóstico da lesão colônica é realizado já na fase maligna, em torno dos 45 anos de idade, com preferência para o lado direito do cólon. Pode haver associação com tumores de endométrio na mulher, estômago, pâncreas, entre outros. É causada por mutação em genes de reparo do DNA (hMSH2, hMLH1, hPMS1, hPMS2, hMSH6/GTBP). A colectomia total deve ser realizada em pacientes com câncer de cólon e HNPCC. Se o tumor estiver localizado no reto, a proctocolectomia total pode ser uma opção. Em indivíduos portadores do defeito genético predisponente ao HNPCC, porém, assintomáticos, a indicação de cirurgias profiláticas é controversa. Atualmente, podem-se identificar indivíduos portadores de defeito genético herdado tanto na FAP como no HNPCC. Esses testes baseiam-se no estudo direto dos genes responsáveis pela respectiva doença ou pela proteína produto dos mesmos. É de suma importância uma abordagem multidisciplinar de pacientes portadores de FAP ou HNPCC, pois existe uma preocupação ética muito grande na realização dos testes genéticos de predisposição, considerando suas conseqüências psicológicas e sociais.

CFH Y402H polymorphism and response to intravitreal Ranibizumab in brazilian patients with neovascular age-related macular degeneration

Objective: To investigate the association between CFH gene polymorphism and response to ranibizumab in Brazilian patients with neovascular age-related macular degeneration (AMD).Methods: 95 patients were genotyped for the CFH rs1061170 (Y402H) single nucleotide polymorphism. Patients with neovascular AMD initially received intravitreal ranibizumab injections for three months and were retreated as needed. Visual acuity (VA) and central retinal thickness (CRT) were measured before treatment and at 1, 3, 6, and 12 months post-treatment.Results: For patients with the TT and TC genotypes, paired comparisons of VA showed a statistically significant improvement when the data obtained at all visits were compared with baseline. Patients homozygous for the risk genotype (CC) did not show a statistically significant improvement when VA obtained at visits 1, 3, 6 and 12 were compared with baseline. For all genotypes, paired comparisons of CRT showed a statistically significant improvement when the data obtained at visits 1, 3, 6 and 12 were compared with baseline.Conclusion: Patients with the CC genotype showed poorer long-term functional response to intravitreal ranibizumab.

Construction and validation of a surgical skills assessment tool for general surgery residency program

Objective: To develop and validate an instrument for measuring the acquisition of technical skills in conducting operations of increasing difficulty for use in General Surgery Residency (GSR) programs. Methods: we built a surgical skills assessment tool containing 11 operations in increasing levels of difficulty. For instrument validation we used the face validaity method. Through an electronic survey tool (Survey MonKey(r)) we sent a questionnaire to Full and Emeritus members of the Brazilian College of Surgeons - CBC - all bearers of the CBC Specialist Title. Results: Of the 307 questionnaires sent we received 100 responses. For the analysis of the data collected we used the Cronbach's alpha test. We observed that, in general, the overall alpha presented with values near or greater than 0.70, meaning good consistency to assess their points of interest. Conclusion: The evaluation instrument built was validated and can be used as a method of assessment of technical skill acquisition in the General Surgery Residency programs in Brazil.

Enfermidades determinadas pelo princípio radiomimético de Pteridium aquilinum (Polypodiaceae)

Por meio de revisão da literatura pertinente foram coligidos e são apresentados os principais dados relativos aos aspectos epidemiológicos, toxicológicos, clínicos, anátomo e histopatológicos observados nos casos de intoxicação pelo princípio radiomimético de Pteridium aquilinum (L.) Kuhn. São abordados a Hematúria Enzoótica Bovina (HEB), os Carcinomas das Vias Digestivas Superiores (CVDS), a Diátese Hemorrágica (DH), os Tumores Intestinais (TI) e a Degeneração Progressiva da Retina (DPR), com ênfase proporcional à importância de cada entidade. Esse estudo objetiva chamar a atenção para o especial significado dessa planta para a pecuária, em função dos prejuízos econômicos por ela determinados e, também, pelo provável risco que ela representa para a saúde humana.