Evaluating Estradiol as a Novel Therapy for Dry Age-Related Macular Degeneration/Geographic Atrophy

Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly population in the United States and is more prevalent in post-menopausal females than age-matched males. There are no current FDA-approved therapies for dry AMD, which composes 80- 90% of all AMD cases. Late stage dry AMD (geographic atrophy) is characterized by extensive loss of the retinal pigment epithelium (RPE) along with degeneration of the photoreceptors, which cause significant central vision loss. AMD is characterized by drusen, which are extracellular deposits located beneath the retina. Drusen contain proteins associated with the process of inflammation such as activated complement proteins, fragments of the membrane attack complex, vitronectin, cholesterol, C-reactive protein, and amyloid-β. Amyloid-β is a cytotoxic protein associated with various neurodegenerative diseases and is commonly found in plaques of Alzheimer’s disease. Based on these findings, we hypothesized that estradiol may have protective effects against the cytotoxic properties of amyloid-β in RPE cells in vitro

Stressful events and adolescent psychopathology: A person-centred approach to expanding adverse childhood experience categories

Stress from cumulative adverse childhood experiences (ACEs) can pose a serious risk of experiencing anxiety, depression, and other mood disorders in adolescence. However, there is a paucity of research identifying specific profiles or combinations of exposure to other forms of stressful life events and their impact on adolescent psychopathology. This study attempted a conceptual expansion of the ACE checklist by examining these stressful events. The study used cross-sectional data from a modified version of the CASE Study survey where 864 adolescents (56% female, n = 480), aged from 11 – 18 years were recruited from four post-primary schools in the North-West region of NI. Latent class analysis of the 20-item stressful events checklist revealed 3 distinct risk classes: a low-risk class (53.5%), at-risk class (42.7%), and an immediate-risk class (3.8%). Results showed those at most risk of adolescent psychopathology had the highest probability of encountering interpersonal relationship issues, experiencing family dysfunction, and having close friends experiencing psychological difficulties. Findings indicate that the original ten ACE categories may be too narrow in focus and do not capture the wide range of childhood adversity. Expanding the ACE checklist to include other stressful events is discussed as these may also be antecedents to psychopathologic responses

Monitoring the production of AAV vectors in insect cells by fluorescence spectroscopy

Adeno-associated viruses (AAV) are among the most promising viral vectors for gene therapy, since they can transduce non-dividing cells from several tissues while maintaining a long-term gene expression. Besides, AAVs possess low immunogenicity compared to other viral vectors, and are physically resistant, which makes them resilient to industrial manufacturing conditions, long-term storage, and in vivo administration. One of the systems available for large scale production of AAVs is the insect cell-baculovirus expression vector system (IC-BEVS). Insect cells grow in suspension to high cell densities with modest growth requirements and without the need of serum supplementation. Consequently, scaling up the production in order to achieve the large number of AAV needed for clinical trials is more straight‑forward than with transfection-based systems. However, methods for online monitoring of AAV production are still lacking. Such methods would allow determination of the best time of harvest in real-time, thus allowing recovery of AAV as soon as its concentration medium was higher. Here we apply Fluorescence Spectroscopy to baculovirus-infected insect cell cultures producing adeno‑associated virus vectors, correlating the spectra to critical process parameters like cell concentration, viability and AAV concentration. Sf9 cells were co-infected with two baculovirus (expressing AAV rep and cap and a CMV-GFP transgene) at low or high multiplicities of infection (MOI), and the culture was followed by Fluorescence Spectroscopy in situ through a bioreactor probe. After an exploratory calibration using data from only one bioreactor, we attested the aptitude of this technique to capture overall data trend: using a 3 component PLS model, we have obtained a calibration NRMSE of 2.9% for total AAV particles per cell, 5.9% for viable cell density and 0.9% for viability). Additional bioreactor productions using different infection parameters (CCI, MOI, time of infection) allowed testing the robustness of fluorescence monitoring to process variability. With this dataset, we tested several pre-treatment methods for the raw spectra, as well as different regression algorithms in order to establish a good predictive model. Ultimately, fluorescence spectroscopy provides a simple tool for online monitoring of key process variables in baculovirus-infected insect cell cultures. Acknowledgments: Funding from Fundação para a Ciência e a Tecnologia, projects EXPL/BBBBIO/1129/2013 and Daniel Pais’ PhD research grant PD/BD/105873/2014