Analisi delle proprietà immunologiche e carcinogeniche di Epstein-Barr Nuclear Antigen 1

The Epstein Barr virus (EBV) is a widespread γ-herpesvirus, more than the 90% of the human population is infected. The first contact with the virus usually happens during the childhood and results in an asymptomatic infection that last for all the life of individual establishing life-long infections in memory B-cells, however might also have been implicated in the pathogenesis of a broad variety of human malignancies including Burkitt’s lymphoma (BL), Hodgkin’s Disease (HD), nasopharyngeal carcinoma (NPC) and post-transplant lymphoproliferative disease (PTLD) Different latency programs lead to different pattern of expression of the EBV latent antigens. The Epstein-Barr virus nuclear antigen 1 is the only antigen expressed in all the EBV-associated tumors (EBNA1). The main topic of the study is the EBNA1 protein, and we wanted to focus on two different aspects that characterize the protein: the immunogenicity and the carcinogenicity. In the first work we identified a new EBNA1-derived CTLs epitope, defined as LQTHIFAEV, that binds the HLA-A2 molecules. Altough the presence of the CTL LQT-restricted any specific killing of targets expressing the native EBNA1 protein was detected. The data, however, represent a new insight about the EBNA1 immunogenicity since a new MHC class I epitope has been discovered. It may also represent a new starting point to investigate the lacking of degradation of EBNA1 and the mechanisms that should be modulated in order to increase its immunogenicity. In the second study we show that EBNA-1 induces chromosomal aberrations and DNA double-strand breaks. These signs of genomic instability are associated with an higher production of reactive oxygen species (ROS). The catalytic subunit of the NADPH oxidase, NOX2/gp91phox, is transcriptionally activated by EBNA-1, while inactivation of the enzyme by chemical inhibitors or RNAi halts ROS production and DDR. These findings highlight a novel function of EBNA-1 that may explain its capacity to promote B-cell immortalization and malignant transformation

High Avidity Binding to DNA Protects Ubiquitylated Substrates from Proteasomal Degradation*

Protein domains that act as degradation and stabilization signals regulate the rate of turnover of proteasomal substrates. Here we report that the bipartite Gly-Arg repeat of the Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 acts as a stabilization signal that inhibits proteasomal degradation in the nucleus by promoting binding to cellular DNA. Protection can be transferred by grafting the domain to unrelated proteasomal substrates and does not involve changes of ubiquitylation. Protection is also afforded by other protein domains that, similar to the Gly-Arg repeat, mediate high avidity binding to DNA, as exemplified by resistance to detergent extraction. Our findings identify high avidity binding to DNA as a portable inhibitory signal that counteracts proteasomal degradation

Characterization of an human leucocyte antigen A2-restricted Epstein–Barr virus nuclear antigen-1-derived cytotoxic T-lymphocyte epitope

The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is regularly expressed in all proliferating virus-infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte antigen (HLA) -A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1-specific cytotoxic T-lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT-specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA-A2-restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT-specific CTL clones did not lyse EBV-carrying lymphoblastoid cell lines and Burkitt’s lymphoma cell lines nor EBNA1-transfected Burkitt’s lymphoma cells but specifically released interferon-γ upon stimulation with HLA-matched EBNA1-expressing cells and this response was enhanced by deletion of the Gly-Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope

MISURE DI COEFFICIENTE DI ASSORBIMENTO IN CAMERA RIVERBERANTE: TEST INTER-LABORATORIO

La ISO 354:2003, ovvero la procedura riconosciuta a livello internazionale per la misurazione dei coefficienti di assorbimento acustico in condizioni di campo diffuso, è attualmente in fase di revisione. Il motivo principale di questa revisione è la limitata riproducibilità dei coefficienti di assorbimento misurati in laboratori diversi. Una rete di laboratori italiani si è unita per svolgere un test inter-laboratorio (ILT) per valutare e confrontare le incertezze di misura risultanti dall'applicazione dell'attuale versione della ISO 354:2003 e della nuova ISO/CD 354:2019. La memoria presenta i risultati delle misurazioni, relativi solamente alla norma in vigore, e fornisce una stima dell'incertezza e dell'accuratezza delle misurazioni attuali

<i>In Vitro</i> Systems Toxicology Assessment of a Candidate Modified Risk Tobacco Product Shows Reduced Toxicity Compared to That of a Conventional Cigarette

Cigarette smoke increases the risk for respiratory and other diseases. Although smoking prevalence has declined over the years, millions of adults choose to continue to smoke. Modified risk tobacco products (MRTPs) are potentially valuable tools for adult smokers that are unwilling to quit their habit. Here, we investigated the biological impact of a candidate MRTP, the tobacco-heating system (THS) 2.2, compared to that of the 3R4F reference cigarette in normal primary human bronchial epithelial cells. Chemical characterization of the THS 2.2 aerosol showed reduced levels of harmful constituents compared to those of a combustible cigarette. Multiparametric indicators of cellular toxicity were measured via real-time cellular analysis and high-content screening. The study was complemented by a whole transcriptome analysis, followed by computational approaches to identify and quantify perturbed molecular pathways. Exposure of cells to 3R4F cigarette smoke resulted in a dose-dependent response in most toxicity end points. Moreover, we found a significant level of perturbation in multiple biological pathways, particularly in those related to cellular stress. By contrast, exposure to THS 2.2 resulted in an overall lower biological impact. At 3R4F doses, no toxic effects were observed. A toxic response was observed for THS 2.2 in some functional end points, but the responses occurred at doses between 3 and 15 times higher than those of 3R4F. The level of biological network perturbation was also significantly reduced following THS 2.2 aerosol exposure compared to that of 3R4F cigarette smoke. Taken together, the data suggest that THS 2.2 aerosol is less toxic than combustible cigarette smoke and thus may have the potential to reduce the risk for smoke-related diseases

A framework for <i>in vitro</i> systems toxicology assessment of e-liquids

<p>Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an <i>in vitro</i> systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air–liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust <i>in vitro</i> assessment of e-liquids and their aerosols.</p