Inauguration of the Centro 3R for the promotion of 3Rs principles in teaching and research

The first European interuniversity center dedicated to promoting 3Rs principles in teaching and research was inaugurated in Pisa, Italy on March 14, 2018. The Centro 3R1 was spearheaded by the Universities of Pisa and Genova. Membership is open to all Italian universities and agreements for twinning across Europe and other countries are being pursued

Teaching and training in substitutive approaches to animal testing: Commitment of the University of Genoa

The 21st century life science requires that scientists become aware of scientific and ethical issues of substitutive approaches to animal testing. For more than 20 years Dr Bassi Team at University of Genoa promotes several activities in Academia on the Replacement of Animal Testing: 2 days training course, lectures within teaching programs, work experience opportunities for graduated or graduating students, stages for Biology, Medicine and Surgery degrees and PhD courses on human in vitro model disease projects, This effort is now improved by Centro3R activities to increase the number of scientists aware of effective alternatives to classical in vivo approaches, and prevent needless suffering of animals

Give meaning to alternative methods to animal testing.

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Assessment of an in vitro physiological relevant model to check therapeutic strategies for glaucoma

Glaucoma is a chronic, progressive and heterogeneous optic neuropathy which affects in the early stage the peripheral vision and then the central vision, leading to irreversible blindness. As known, in glaucoma the trabecular meshwork represent the main tissue which is impaired by chronic oxidative stress, aging and increase of intraocular pressure. Today, the lack of human-based models, with characteristics of high repeatability and reproducibility as well, called for an high-quality in vitro model with a good degree of resemblance for the tissue or organ of interest as a basis for new drug testing. Our team has been committed to this purpose by assessment of an in vitro 3D TM human-based model, closer to in vivo, using millifluidic technology, to better identify the key events underlying the pathogenesis of glaucoma and to evaluate new therapies targeted at disease treatment and prevention

2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study

A physiologically relevant in vitro human-based model could be the \u2018gold standard\u2019 to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D

Simulated microgravity induces nuclear translocation of Bax and BCL-2 in glial cultured C6 cells

Alterations in the control of apoptotic processes were observed in cells during space flight or under simulated microgravity, the latter obtained with the 3D-Random Positioning Machine (3D-RPM). Usually the proteins Bax and Bcl-2, act as pro- or anti-apoptotic regulators. Here we investigated the effects of simulated microgravity obtained by the 3D-RPM on cell viability, localization and expression of Bax and Bcl-2 in cultures of glial cancerous cells. We observed for the first time a transient cytoplasmic/nuclear translocation of Bax and Bcl-2 triggered by changing gravity vector. Bax translocates into the nucleus after 1 h, is present simultaneously in the cytoplasm after 6 h and comes back to the cytoplasm after 24 h. Bcl-2 translocate into the nucleus only after 6 h and comes back to the cytoplasm after 24 h. Physiological meaning, on the regulation of apoptotic event and possible applicative outcomes of such finding are discussed

Short-term and long-term effects of tibolone in postmenopausal women

Background: Tibolone is a synthetic steroid used for the treatment of menopausal symptoms, on the basis of short-term data suggesting its efficacy. We considered the balance between the benefits and risks of tibolone. Objectives: To evaluate the effectiveness and safety of tibolone for treatment of postmenopausal and perimenopausal women. Search methods: In October 2015, we searched the Gynaecology and Fertility Group (CGF) Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase and PsycINFO (from inception), the Cumulative Index to Nursing and Allied Health Literature (CINAHL) and clinicaltrials.gov. We checked the reference lists in articles retrieved. Selection criteria: We included randomised controlled trials (RCTs) comparing tibolone versus placebo, oestrogens and/or combined hormone therapy (HT) in postmenopausal and perimenopausal women. Data collection and analysis: We used standard methodological procedures of The Cochrane Collaboration. Primary outcomes were vasomotor symptoms, unscheduled vaginal bleeding and long-term adverse events. We evaluated safety outcomes and bleeding in studies including women either with or without menopausal symptoms. Main results: We included 46 RCTs (19,976 women). Most RCTs evaluated tibolone for treating menopausal vasomotor symptoms. Some had other objectives, such as assessment of bleeding patterns, endometrial safety, bone health, sexuality and safety in women with a history of breast cancer. Two included women with uterine leiomyoma or lupus erythematosus. Tibolone versus placebo Vasomotor symptoms Tibolone was more effective than placebo (standard mean difference (SMD) -0.99, 95% confidence interval (CI) -1.10 to -0.89; seven RCTs; 1657 women; moderate-quality evidence), but removing trials at high risk of attrition bias attenuated this effect (SMD -0.61, 95% CI -0.73 to -0.49; odds ratio (OR) 0.33, 85% CI 0.27 to 0.41). This suggests that if 67% of women taking placebo experience vasomotor symptoms, between 35% and 45% of women taking tibolone will do so. Unscheduled bleeding Tibolone was associated with greater likelihood of bleeding (OR 2.79, 95% CI 2.10 to 3.70; nine RCTs; 7814 women; I2 = 43%; moderate-quality evidence). This suggests that if 18% of women taking placebo experience unscheduled bleeding, between 31% and 44% of women taking tibolone will do so. Long-term adverse events Most of the studies reporting these outcomes provided follow-up of two to three years (range three months to three years). Breast cancer We found no evidence of differences between groups among women with no history of breast cancer (OR 0.52, 95% CI 0.21 to 1.25; four RCTs; 5500 women; I2= 17%; very low-quality evidence). Among women with a history of breast cancer, tibolone was associated with increased risk (OR 1.5, 95% CI 1.21 to 1.85; two RCTs; 3165 women; moderate-quality evidence). Cerebrovascular events We found no conclusive evidence of differences between groups in cerebrovascular events (OR 1.74, 95% CI 0.99 to 3.04; four RCTs; 7930 women; I2 = 0%; very low-quality evidence). We obtained most data from a single RCT (n = 4506) of osteoporotic women aged 60 to 85 years, which was stopped prematurely for increased risk of stroke. Other outcomes Evidence on other outcomes was of low or very low quality, with no clear evidence of any differences between the groups. Effect estimates were as follows: \u2022 Endometrial cancer: OR 2.04, 95% CI 0.79 to 5.24; nine RCTs; 8504 women; I2 = 0%. \u2022 Cardiovascular events: OR 1.38, 95% CI 0.84 to 2.27; four RCTs; 8401 women; I2 = 0%. \u2022 Venous thromboembolic events: OR 0.85, 95% CI 0.37 to 1.97; 9176 women; I2 = 0%. \u2022 Mortality from any cause: OR 1.06, 95% CI 0.79 to 1.41; four RCTs; 8242 women; I2 = 0%. Tibolone versus combined HT Vasomotor symptoms Combined HT was more effective than tibolone (SMD 0.17, 95% CI 0.06 to 0.28; OR 1.36, 95% CI 1.11 to 1.66; nine studies; 1336 women; moderate-quality evidence). This result was robust to a sensitivity analysis that excluded trials with high risk of attrition bias, suggesting a slightly greater disadvantage of tibolone (SMD 0.25, 95% CI 0.09 to 0.41; OR 1.57, 95% CI 1.18 to 2.10). This suggests that if 7% of women taking combined HT experience vasomotor symptoms, between 8% and 14% of women taking tibolone will do so. Unscheduled bleeding Tibolone was associated with a lower rate of bleeding (OR 0.32, 95% CI 0.24 to 0.41; 16 RCTs; 6438 women; I2 = 72%; moderate-quality evidence). This suggests that if 47% of women taking combined HT experience unscheduled bleeding, between 18% and 27% of women taking tibolone will do so. Long-term adverse events Most studies reporting these outcomes provided follow-up of two to three years (range three months to three years). Evidence was of very low quality, with no clear evidence of any differences between the groups. Effect estimates were as follows: \u2022 Endometrial cancer: OR 1.47, 95% CI 0.23 to 9.33; five RCTs; 3689 women; I2 = 0%. \u2022 Breast cancer: OR 1.69, 95% CI 0.78 to 3.67; five RCTs; 4835 women; I2 = 0%. \u2022 Venous thromboembolic events: OR 0.44, 95% CI 0.09 to 2.14; four RCTs; 4529 women; I2 = 0%. \u2022 Cardiovascular events: OR 0.63, 95% CI 0.24 to 1.66; two RCTs; 3794 women; I2 = 0%. \u2022 Cerebrovascular events: OR 0.76, 95% CI 0.16 to 3.66; four RCTs; 4562 women; I2 = 0%. \u2022 Mortality from any cause: only one event reported (two RCTs; 970 women). Authors' conclusions: Moderate-quality evidence suggests that tibolone is more effective than placebo but less effective than HT in reducing menopausal vasomotor symptoms, and that tibolone is associated with a higher rate of unscheduled bleeding than placebo but with a lower rate than HT. Compared with placebo, tibolone increases recurrent breast cancer rates in women with a history of breast cancer, and may increase stroke rates in women over 60 years of age. No evidence indicates that tibolone increases the risk of other long-term adverse events, or that it differs from HT with respect to long-term safety. Much of the evidence was of low or very low quality. Limitations included high risk of bias and imprecision. Most studies were financed by drug manufacturers or failed to disclose their funding source

Efficacy and Tolerability of Perampanel in Brain Tumor-Related Epilepsy: A Systematic Review

(1) Background: Epilepsy is a frequent comorbidity in patients with brain tumors, in whom seizures are often drug-resistant. Current evidence suggests that excess of glutamatergic activity in the tumor microenvironment may favor epileptogenesis, but also tumor growth and invasiveness. The selective non-competitive -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist perampanel (PER) was demonstrated to be efficacious and well-tolerated in patients with focal seizures. Moreover, preclinical in vitro studies suggested a potential anti-tumor activity of this drug. In this systematic review, the clinical evidence on the efficacy and tolerability of PER in brain tumor-related epilepsy (BTRE) is summarized. (2) Methods: Five databases and two clinical trial registries were searched from inception to December 2022. (3) Results: Seven studies and six clinical trials were included. Sample size ranged from 8 to 36 patients, who received add-on PER (mean dosage from 4 to 7 mg/day) for BTRE. After a 6–12 month follow-up, the responder rate (% of patients achieving seizure freedom or reduction 50% of seizure frequency) ranged from 75% to 95%, with a seizure freedom rate of up to 94%. Regarding tolerability, 11–52% of patients experienced non-severe adverse effects (most frequent: dizziness, vertigo, anxiety, irritability). The retention rate ranged from 56% to 83%. However, only up to 12.5% of patients discontinued the drug because of the adverse events. (4) Conclusions: PER seems to be efficacious, safe, and well-tolerated in patients with BTRE. Further randomized studies should be conducted in more homogeneous and larger populations, also evaluating the effect of PER on tumor progression, overall survival, and progression-free survival

Evidence for the Involvement of Lipid Rafts and Plasma Membrane Sphingolipid Hydrolases in Pseudomonas aeruginosa

Cystic fibrosis (CF) is the most common autosomal genetic recessive disease caused by mutations of gene encoding for the cystic fibrosis transmembrane conductance regulator. Patients with CF display a wide spectrum of symptoms, the most severe being chronic lung infection and inflammation, which lead to onset of cystic fibrosis lung disease. Several studies indicate that sphingolipids play a regulatory role in airway inflammation. The inhibition and downregulation of GBA2, the enzyme catabolizing glucosylceramide to ceramide, are associated with a significant reduction of IL-8 production in CF bronchial epithelial cells. Herein, we demonstrate that GBA2 plays a role in the proinflammatory state characterizing CF cells. We also report for the first time that Pseudomonas aeruginosa infection causes a recruitment of plasma membrane-associated glycosphingolipid hydrolases into lipid rafts of CuFi-1-infected cells. This reorganization of cell membrane may be responsible for activation of a signaling cascade, culminating in aberrant inflammatory response in CF bronchial epithelial cells upon bacterial infection. Taken together, the presented data further support the role of sphingolipids and their metabolic enzymes in controlling the inflammatory response in CF

GM1 as adjuvant of innovative therapies for cystic fibrosis disease

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function