35 research outputs found
MODULAZIONE DEI FENOMENI DI SENESCENZA CELLULARE DI ADIPOCITI E PREADIPOCITI IN VITRO
No full textL’invecchiamento è accompagnato da una complessa rete di processi biologici caratterizzati da uno stato infiammatorio cronico basale, a sua volta correlato allo sviluppo di patologie età-associate come: aterosclerosi, osteoartriti, Alzheimer, diabete di tipo 2. Una sempre più approfondita analisi del processo di senescenza tessutospecifico potrebbe consentire l’individuazione di target terapeutici utili a modulare il processo complessivo dell’invecchiamento stesso. L’invecchiamento è causa di declino delle funzionalità sistemiche e il tessuto adiposo è uno degli organi maggiormente colpiti in quanto subisce cambiamenti significativi che riguardano la quantità, la distribuzione, la composizione cellulare e l’attività endocrina, giocando un ruolo chiave nell’insorgenza di insulino-resistenza, di disfunzioni metaboliche e infiammazione sistemica. L’abbondanza del tessuto adiposo e le diverse funzioni da esso esercitate lo rendono un organo fondamentale per la comprensione della fisiologia dell’intero organismo. Tuttavia, i cambiamenti a cui è sottoposto durante il processo di invecchiamento sono solo in parte noti e oggetto di studio negli ultimi anni. È quindi fondamentale conoscere i processi molecolari che stanno alla base della fisiopatologia del tessuto adiposo e del suo invecchiamento in modo da poter individuare strategie terapeutiche per le malattie correlate all’invecchiamento. In questo studio, è stato messo a punto un modello cellulare di senescenza in vitro attraverso il trattamento con perossido di idrogeno (H2O2) in adipociti e preadipociti 3T3-L1 murini. Successivamente è stata valutata la potenziale attività senolitica della quercetina esaminandone l’effetto antiossidante e antinfiammatorio, cercando di individuarne il target molecolare.Aging is associated with pathological age-related processes as inflammatory low-grade state, atherosclerosis, Alzheimer's disease, type II diabetes and osteoarthritis diseases. A deep analysis of the tissue-specific senescence process could allow the identification of therapeutic targets to modulate the aging process itself. In this study, premature senescence model was developed in vitro through treatment with oxygen peroxide (H2O2) in murine 3T3-L1 preadipocytes before and after induction to mature adipocytes. H2O2 treated cells showed characteristic senescence-associated features including senescence-associated beta-galactosidase activity (SA-ß-gal), activation of reactive oxygen species (ROS) development of senescence-associated secretory phenotype (SASP), induction of cell cycle inhibitors P-21 as well as induction of pro-inflammatory transduction factor NFκB and a downregulation of deacetylase SIRT1. We found that stimulation with H2O2 results in an induction of miR-155-5p expression in preadipocytes and in mature adipocytes. The treatment with quercetin (20 μM) showed significant decrease in the number of ß-galactosidase positive cell along with the suppression of ROS, NFκB and SASP factor in both cell models. In addition, quercetin treatment also upregulated protein expression of SIRT1 and decreased miR-155-5p expression. These results suggest that quercetin acts as a potential senolytic agent in both preadipocytes and adipocytes cell models, inhibiting ROS production and proinflammatory miR-155-5p
In vitro model of chronological aging of adipocytes: Interrelationships with hypoxia and oxidation
No full textAging is a physiological process characterized by an age-progressive decline in intrinsic physiological functions, with an increased risk of developing chronic metabolic conditions, such as insulin resistance and diabetes. Furthermore, from a physiopathological point of view, several authors describe an association between oxidative stress, hypoxia and these metabolic conditions. It had been suggested that adipose tissue (AT) dysfunction, senescent cell accumulation and proinflammatory pathways may be involved in this processes. The purpose of this study was to develop an in vitro model to study the progressive morphological and functional changes of adipocytes with aging, in standard culture conditions and after severe hypoxia and hydrogen peroxide treatment. We evaluated the degree of apoptosis and intracellular reactive oxygen species (ROS) accumulation as well as the gene expression profile of aging adipocytes. Our results show that aged adipocytes become senescent, undergo apoptosis, accumulate ROS, and present an inflammatory profile with an increase in mRNA expression level of key proteins related to the remodeling of the extracellular matrix (ECM). Aged adipocytes present increased levels of p53, p21 and p16, key regulators of senescence, and a decrease in SIRT-1 protein compared to younger cells. Moreover, adipocytes aged in hypoxia or in oxidative stress conditions represent a model of accelerated aging with a decrease in their area, a greater proportion of apoptotic and of intracellular ROS accumulation compared to controls. This study characterizes the progressive morphological and functional changes in aging adipocytes during prolonged cell cultures and explores the addictive effects of hypoxia and oxidation, given at different stages of cellular maturation and senescence
Enforcing surveillance of antimicrobial resistance and antibiotic use to drive stewardship: experience in a paediatric setting
No full textBackground: Antibiotic Stewardship (AS) interventions in paediatrics are still not standardized regarding methodology, metrics, and outcomes. We report the results of an AS intervention in the paediatric area based on education and guideline provision via an electronic App. Materials and methods: The AS intervention was conducted in 2021 through observation, education, audit and feedback and provision of an electronic App (Firstline.org) to support antibiotic prescription based on local susceptibility data. The primary outcome was the antibiotic consumption in the 12-months following the intervention (year 2022) compared to a historical 12-month control (year 2019) via an interrupted time series analysis. Secondary outcomes were appropriateness of therapy, length-of-stay, 30-day readmission, transfers to the paediatric intensive care unit, in-hospital mortality, and prevalence of antimicrobial resistance (AMR). Results: During the post-intervention phase, 29 cross-sectional audits and feedback were conducted including 467 patients. Prescriptions were appropriate according to the guidelines in 85.7% of cases, with a stable trend over time. A significant decrease of antibiotic consumption was measured in terms of Defined Daily Doses per 1000 Patient Days (-222.13; p<0.001) and Days of Therapy per 1000 Patient Days (-452.49; p <0.001) in the post-intervention period with a clear inversion of the Access to Watch ratio (from 0.7 to 1.7). Length of stay, in-hospital mortality, ICU transfers, and incidence of AMR infections remained stable, while 30-day readmission decreased from 4.9 per 100 admissions to 2.8 per 100 admissions (p <0.001). Conclusions: The intervention was associated with a significant reduction in antimicrobial consumption and an increase in the appropriateness of prescriptions. Electronic tools can be of value in promoting adherence to guidelines and ensuring the sustainability of results
Role of adipose tissue in melanoma cancer microenvironment and progression
No full textBackground:An epidemiological association between excess weight and increased risk of cancer has been described in melanoma, for which the physiopathological mechanisms are still unknown. The study of tumor microenvironment and of the role of adipocytes in cancer development, progression and metastasis has recently received great interest. However, the role of peritumoral adipocytes has been characterized only in a few types of cancer, and in melanoma it still remains to be defined.Methods:We investigated the interactions between adipocytes and melanoma cells using an in vitro co-culture system. We studied the morphological and functional properties of 3T3-L1 adipocytes before and after co-culture with A375 melanoma cells, in order to assess the role of adipocytes on melanoma migration.Results:Morphological analysis showed that after 6 days of co-culture 3T3-L1 adipocytes were reduced in number and size. Moreover, we observed the appearance of dedifferentiated cells with a fibroblast-like phenotype that were not present in controls and that had lost the expression of some adipocyte-specific genes, and increased the expression of collagen, metalloproteinases and genes typical of dedifferentiation processes. Through the Matrigel Invasion Test, as well the Scratch Test, it was possible to observe that co-culture with adipocytes induced in melanoma cells increased migratory capacity, as compared with controls. In particular, the increase in migration observed in co-culture was suppressed after adding the protein SFRP-5 in the medium, supporting the involvement of the Wnt5a pathway. The activation of this pathway was further characterized by immunofluorescence and western blot analysis, showing in melanocytes in co-culture the activation of β-catenin and LEF-1, two transcription factors involved in migration processes, neo-angiogenesis and metastasis.Conclusions:These data allow us to hypothesize a dedifferentiation process of adipocytes toward fibroblast-like cells, which can promote migration of melanoma cells through activation of Wnt5a and the intracellular pathways of β-catenin and LEF-1
Senolytic effects of quercetin in an in vitro model of pre-adipocytes and adipocytes induced senescence
No full textThe dysfunction of adipose tissue with aging and the accumulation of senescent cells has been implicated in the pathophysiology of chronic diseases. Recently interventions capable of reducing the burden of senescent cells and in particular the identification of a new class of drugs termed senolytics have been object of extensive investigation. We used an in vitro model of induced senescence by treating both pre-adipocytes as well as mature adipocytes with hydrogen peroxide (H2O2) at a sub-lethal concentration for 3 h for three consecutive days, and hereafter with 20 uM quercetin at a dose that in preliminary experiments resulted to be senolytic without cytotoxicity. H2O2 treated pre-adipocytes and adipocytes showed typical senescence-associated features including increased beta-galactosidase activity (SA-ss-gal) and p21, activation of ROS and increased expression of pro-inflammatory cytokines. The treatment with quercetin in senescent pre-adipocytes and adipocytes was associated to a significant decrease in the number of the SA-beta-gal positive cells along with the suppression of ROS and of inflammatory cytokines. Besides, quercetin treatment decreased miR-155-5p expression in both models, with down-regulation of p65 and a trend toward an up-regulation of SIRT-1 in complete cell extracts. The senolytic compound quercetin could affect AT ageing by reducing senescence, induced in our in vitro model by oxidative stress. The downregulation of miRNA-155-5p, possibly through the modulation of NF-kappa B and SIRT-1, could have a key role in the effects of quercetin on both pre-adipocytes and adipocytes
Pseudomonas aeruginosa reduces the expression of CFTR via post-translational modification of NHERF1
No full textP. aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling.
Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host-cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host–cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds
The antimicrobial resistance travel tool, an interactive evidence-based educational tool to limit antimicrobial resistance spread
No full textBACKGROUND: International travel has been recognized as a risk factor contributing to the spread of antimicrobial resistance (AMR). However, tools focused on AMR in the context of international travel and designed to guide decision-making are limited. We aimed at developing an evidence-based educational tool targeting both healthcare professionals (HCPs) and international travellers to help prevent the spread of AMR. METHODS: A literature review on 12 antimicrobial-resistant bacteria (ARB) listed as critical and high tiers in the WHO Pathogen Priority List covering four key areas was carried out: AMR surveillance data; epidemiological studies reporting ARB prevalence data on carriage in returning travellers; guidance documents reporting indications on screening for ARB in returning travellers and recommendations for ARB prevention for the public. The evidence, catalogued at country-level, provided the content for a series of visualizations that allow assessment of the risk of AMR acquisition through travel. RESULTS: Up to January 2021, the database includes data on: (i) AMR surveillance for 2.018.241 isolates from 86 countries; (ii) ARB prevalence of carriage from 11.679 international travellers and (iii) 15 guidance documents published by major public health agencies. The evidence allowed the development of a consultation scheme for the evaluation of risk factors, prevalence of carriage, proportion and recommendations for screening of AMR. For the public, pre-travel practical measures to minimize the risk of transmission were framed. CONCLUSIONS: This easy-to-use, annually updated, freely accessible AMR travel tool (https://epi-net.eu/travel-tool/overview/), is the first of its kind to be developed. For HCPs, it can provide a valuable resource for teaching and a repository that facilitates a stepwise assessment of the risk of AMR spread and strengthen implementation of optimized infection control measures. Similarly, for travellers, the tool has the potential to raise awareness of AMR and outlines preventive measures that reduce the risk of AMR acquisition and spread
Diagnostic Accuracy of a Rapid SARS-CoV-2 Antigen Test Among People Experiencing Homelessness: A Prospective Cohort and Implementation Study
No full textIntroduction: Detection strategies in vulnerable populations such as people experiencing homelessness (PEH) need to be explored to promptly recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks. This study investigated the diagnostic accuracy of a rapid SARS-CoV-2 Ag test in PEH during two pandemic waves compared with gold standard real-time multiplex reverse transcription polymerase chain reaction (rtRT-PCR). Methods: All PEH ≥ 18 years requesting residence at the available shelters in Verona, Italy, across two cold-weather emergency periods (November 2020-May 2021 and December 2021-April 2022) were prospectively screened for SARS-CoV-2 infection by means of a naso-pharyingeal swab. A lateral flow immunochromatographic assay (Biocredit® COVID-19 Ag) was used as antigen-detecting rapid diagnostic test (Ag-RDT). The rtRT-PCR was performed with AllplexTM SARS-CoV-2 assay kit (Seegene). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated as measures for diagnostic accuracy. Results: Overall, 503 participants were enrolled during the two intervention periods for a total of 732 paired swabs collected: 541 swabs in the first period and 191 in the second. No significant differences in demographic and infection-related characteristics were observed in tested subjects in the study periods, except for the rate of previous infection (0.8% versus 8%; p < 0.001) and vaccination (6% versus 73%; p < 0.001). The prevalence of SARS-CoV-2 in the cohort was 8% (58/732 swabs positive with rtRT-PCR). Seventeen swabs were collected from symptomatic patients (7%). Among them, the concordance between rtRT-PCR and Ag-RDT was 100%, 7 (41.2%) positive and 10 negative pairs. The overall sensitivity of Ag-RDT was 63.8% (95% CI 60.3-67.3) and specificity was 99.8% (95% CI 99.6-100). PPV and NPV were 97.5% and 96.8%, respectively. Sensitivity and specificity did not change substantially across the two periods (65.1% and 99.8% in 2020-2021 vs. 60% and 100% in 2021-2022). Conclusions: A periodic Ag-RDT-based screening approach for PEH at point of care could guide preventive measures, including prompt isolation, without referral to hospital-based laboratories for molecular test confirmation in case of positive detection even in individuals asymptomatic for COVID-19. This could help reduce the risk of outbreaks in shelter facilities
The Emerging Resistance Index: tracking early resistance to new antibiotics
No full text: The high clinical burden of antimicrobial resistance (AMR) and the dwindling antibiotic pipeline demand standardised tools to monitor resistance development to new antibiotics. The Emerging Resistance Index (ERI) is a novel quantitative tool designed to track resistance to newly developed antibiotics. The ERI incorporates various parameters: AMR pooled proportions and trends, availability of AMR and antimicrobial consumption surveillance systems, time lag between antibiotic approval and resistance detection, and the average annual number of outbreaks caused by resistant strains. AMR and antimicrobial consumption data on 13 new antibiotics were extracted by reviewing the literature and the reports from 38 surveillance systems included in the European Clinical Research Alliance on Infectious Diseases-The Epidemiology Network central data repository, encompassing 27 EU countries, four European Free Trade Association countries, and the UK. Our analysis revealed rapid resistance development to all new Gram-negative-targeting antibiotics, with particularly high ERI values for imipenem-relebactam and cefiderocol. Combinations of old β-lactams with novel inhibitors were especially prone to rapid resistance development. The ERI could offer a quantitative and dynamic tool for monitoring resistance trends. Also, the ERI could support more timely surveillance efforts, inform antibiotic policy decisions, and aid in prioritising antibiotic research and development
INVOLVEMEMENT OF GBA2 IN THE INFLAMMATORY RESPONSE TO PSEUDOMONAS AERUGINOSA IN CYSTIC FIBROSIS
No full textCystic fibrosis (CF) lung disease is characterized by progressive chronic infection and inflammation of the airways representing the major cause of mortality in patients. Current anti-inflammatory strategies for the treatment of pulmonary disease in CF are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Several pieces of evidences highlight the role of sphingolipids (SLs) in CF. In fact, ceramide accumulation has been demonstrated in lower airway of CF patients (Brodlie M, et al. Am J Respir Crit Care Med. 2010;182:369-75). Moreover, the infection of bronchial epithelial cells with Pseudomonas aeruginosa (PAO) activated host acid sphingomyelinase leading to ceramide generation at the cell surface, which regulates the release of inflammatory cytokines, as IL-8 (Grassmé H, et al. Nat Med. 2003;9(3):322-30). In addition, miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by PAO and restores F508del-CFTR chloride channel activity even if the molecular mechanism involved is unknown so far (Loberto N, et al. PLoS One. 2014;9:e104763). Taking into account this evidence, the aim of our study was to investigate the molecular mechanism linking GBA2 and the inflammatory response in CF cells. Our experiments were performed on both a human CF bronchial epithelial cell line as well as in human primary bronchial cells. Using these experimental models, we found that the major part of the ceramide produced at the cell plasma membrane level of CF bronchial epithelial cells, subjected to PAO infection, derives from the action of GBA2. These data were also confirmed by the use of a potent inhibitor of GBA2, N-(5adamantane-1-yl-methoxy)pentyl)- deoxynojirimycin (Genz529648). We also examined the impact of lowering the expression of GBA2 in human CF bronchial epithelial cells exposed to P. aeruginosa using siRNA oligonucleotides. Interestingly, we found that basal IL-8 expression and release is reduced in both uninfected and infected cells by PAO. In conclusion, our study proposes GBA2 as a novel target to reduce the pro-inflammatory response to PAO in CF bronchial cells. These results further support the use of modulators of SL metabolism to treat CF lung inflammation
