Retrospective Assessment of Animals Experimentation Projects in Romania – A Critical Analysis of Non-Technical Summaries

The aim of the present inquiry was to design an overview about the non-technical summaries available on the web page of the National Sanitary Veterinary and Food Safety Agency (Romania). We analyzed the compliance to the requirement of replacement, reduction and refinement (the 3R’s). 56 projects were found, the most of them (48), were assigned as “basic research”, 2 as “translational and applied research”, 2 as “regulatory use and routine analysis”, while 4 projects targeted “higher education or training”. The most common animals species used were rats, and mice, followed by rabbits, pigs, hamsters, guinea pigs and frogs (Rana sp.). According to cumulative severity of the project, 20 projects were classified as “severe”, six as “moderate”, three “mild”, and three as “non-recovery”. 24 projects had nor a severity assessment, neither a proper description of the level of severity. In our opinion, the number of projects classified as “severe” is too high; at least for some of them, the level of severity could be reduced using appropriate pain control techniques and / or human end-points. Overall, our recommendation is further improving the knowledge and skills of the personal involved in authorization and execution of the projects and of the authority inspectors that authorized the projects

Increased Degradation Rates in the Components of the Mitochondrial Oxidative Phosphorylation Chain in the Cerebellum of Old Mice

Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy. Thus, an imbalance in mitochondrial turnover can contribute to the diminution of cellular function seen during aging. Mitochondrial biogenesis and mitophagy are mediated by a set of proteins including MFN1, MFN2, OPA1, DRP1, FIS1 as well as DMN1l and DNM1, all of which are required for mitochondrial fission. Using N15 labeling, we report that the turnover rates for DMN1l and FIS1 go in opposite directions in the cerebellum of 22-month-old C57BL6j mice as compared to 3-month-old mice. Previous studies have reported decreased turnover rates for the mitochondrial respiratory complexes of aged rodents. In contrast, we found increased turnover rates for mitochondrial proteins of the oxidative phosphorylation chain in the aged mice as compared to young mice. Furthermore, the turnover rate of the components that are most affected by aging –complex III components (ubiquinol cytochrome C oxidoreductase) and complex IV components (cytochrome C oxidase)– was significantly increased in the senescent cerebellum. However, the turnover rates of proteins involved in mitophagy (i.e., the proteasomal and lysosomal degradation of damaged mitochondria) were not significantly altered with age. Overall, our results suggest that an age-related imbalance in the turnover rates of proteins involved in mitochondrial biogenesis and mitophagy (DMN1l, FIS1) in conjunction with an age-related imbalance in the turnover rates of proteins of the complexes III and IV of the electron transfer chain, might impair cerebellar mitochondrial bioenergetics in old mice

Table_1.XLSX

<p>Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy. Thus, an imbalance in mitochondrial turnover can contribute to the diminution of cellular function seen during aging. Mitochondrial biogenesis and mitophagy are mediated by a set of proteins including MFN1, MFN2, OPA1, DRP1, FIS1 as well as DMN1l and DNM1, all of which are required for mitochondrial fission. Using N15 labeling, we report that the turnover rates for DMN1l and FIS1 go in opposite directions in the cerebellum of 22-month-old C57BL6j mice as compared to 3-month-old mice. Previous studies have reported decreased turnover rates for the mitochondrial respiratory complexes of aged rodents. In contrast, we found increased turnover rates for mitochondrial proteins of the oxidative phosphorylation chain in the aged mice as compared to young mice. Furthermore, the turnover rate of the components that are most affected by aging –complex III components (ubiquinol cytochrome C oxidoreductase) and complex IV components (cytochrome C oxidase)– was significantly increased in the senescent cerebellum. However, the turnover rates of proteins involved in mitophagy (i.e., the proteasomal and lysosomal degradation of damaged mitochondria) were not significantly altered with age. Overall, our results suggest that an age-related imbalance in the turnover rates of proteins involved in mitochondrial biogenesis and mitophagy (DMN1l, FIS1) in conjunction with an age-related imbalance in the turnover rates of proteins of the complexes III and IV of the electron transfer chain, might impair cerebellar mitochondrial bioenergetics in old mice.</p

Table_2.XLSX

<p>Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy. Thus, an imbalance in mitochondrial turnover can contribute to the diminution of cellular function seen during aging. Mitochondrial biogenesis and mitophagy are mediated by a set of proteins including MFN1, MFN2, OPA1, DRP1, FIS1 as well as DMN1l and DNM1, all of which are required for mitochondrial fission. Using N15 labeling, we report that the turnover rates for DMN1l and FIS1 go in opposite directions in the cerebellum of 22-month-old C57BL6j mice as compared to 3-month-old mice. Previous studies have reported decreased turnover rates for the mitochondrial respiratory complexes of aged rodents. In contrast, we found increased turnover rates for mitochondrial proteins of the oxidative phosphorylation chain in the aged mice as compared to young mice. Furthermore, the turnover rate of the components that are most affected by aging –complex III components (ubiquinol cytochrome C oxidoreductase) and complex IV components (cytochrome C oxidase)– was significantly increased in the senescent cerebellum. However, the turnover rates of proteins involved in mitophagy (i.e., the proteasomal and lysosomal degradation of damaged mitochondria) were not significantly altered with age. Overall, our results suggest that an age-related imbalance in the turnover rates of proteins involved in mitochondrial biogenesis and mitophagy (DMN1l, FIS1) in conjunction with an age-related imbalance in the turnover rates of proteins of the complexes III and IV of the electron transfer chain, might impair cerebellar mitochondrial bioenergetics in old mice.</p

Technology transfer of oil-in-water emulsion adjuvant manufacturing for pandemic influenza vaccine production in Romania: preclinical evaluation of split virion inactivated H5N1 vaccine with adjuvant

Millions of seasonal and pandemic influenza vaccine doses containing oil-in-water emulsion adjuvant have been administered in order to enhance and broaden immune responses and to facilitate antigen sparing. Despite the enactment of a Global Action Plan for Influenza Vaccines and a multi-fold increase in production capabilities over the past 10 years, worldwide capacity for pandemic influenza vaccine production is still limited. In developing countries, where routine influenza vaccination is not fully established, additional measures are needed to ensure adequate supply of pandemic influenza vaccines without dependence on the shipment of aid from other, potentially impacted first-world countries. Adaptation of influenza vaccine and adjuvant technologies by developing country influenza vaccine manufacturers may enable antigen sparing and corresponding increases in global influenza vaccine coverage capacity. Following on previously described work involving the technology transfer of oil-in-water emulsion adjuvant manufacturing to a Romanian vaccine manufacturing institute, we herein describe the preclinical evaluation of inactivated split virion H5N1 influenza vaccine with emulsion adjuvant, including immunogenicity, protection from virus challenge, antigen sparing capacity, and safety. In parallel with the evaluation of the bioactivity of the tech-transferred adjuvant, we also describe the impact of concurrent antigen manufacturing optimization activities. Depending on the vaccine antigen source and manufacturing process, inclusion of adjuvant was shown to enhance and broaden functional antibody titers in mouse and rabbit models, promote protection from homologous virus challenge in ferrets, and facilitate antigen sparing. Besides scientific findings, the operational lessons learned are delineated in order to facilitate adaptation of adjuvant technologies by other developing country institutes to enhance global pandemic influenza preparedness