Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

Lentiviral (LV) vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. In this study, we evaluated the transduction efficiency of a last-generation LV vector bearing Green Fluorescent Protein (GFP) gene as well as cytotoxicity and tight junction (TJ) integrity in a polarized model of airway epithelial cells. High multiplicities of infection (MOI) showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (TER) and delocalization of occludin from the TJs. To increase LV efficiency at low LV:cell ratio, we employed noncovalent association with the polycation branched 25 kDa polyethylenimine (PEI). Transduction of cells with PEI/LV particles resulted in 2.5–3.6-fold increase of percentage of GFP-positive cells only at the highest PEI:LV ratios (1×107 PEI molecules/transducing units with 50 MOI LV) as compared to plain LV. At this dose PEI/LV transduction resulted in 6.5 ± 2.4% of propidium iodide-positive cells. On the other hand, PEI/LV particles did not determine any alteration of TER and occludin localization. We conclude that PEI may be useful for improving the efficiency of gene transfer mediated by LV vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness

Nonischemic left ventricular scar and cardiac sudden death in the young

Nonischemic Left Ventricular Scar (NLVS) is a pattern of myocardial injury characterized by midventricular and/or subepicardial gadolinium hyper enhancement at cardiac magnetic resonance, in absence of significant coronary artery disease. We aimed to evaluate the prevalence of NLVS in juvenile sudden cardiac death and to ascertain its aetiology at autopsy. We examined 281 consecutive cases of sudden death of subjects aged 1 to 35 years of age. NLVS was defined as a thin, grey rim of subepicardial and/or midmyocardial scar in the left ventricular free wall and/or the septum, in absence of significant stenosis of coronary arteries. NLVS was the most frequent finding (25%) in sudden deaths occurring during sports. Myocardial scar was localized most frequently within the left ventricular posterior wall, and affected the subepicardial myocardium, often extending to the midventricular layer. On histology it consisted of fibrous or fibro-adipose tissue. Right ventricular involvement was always present. Patchy lymphocytic infiltrates were frequent. Genetic and molecular analyses clarified the aetiology of NLVS in a subset of cases. ECG recordings were available in over half of subjects. The most frequent abnormality was the presence of low QRS voltages (< 0,5 mV) in limb leads. In serial ECG tracings, the decrease in QRS voltages appeared, in some way progressive. NLVS is the most frequent morphologic substrate of juvenile cardiac sudden death in sports. It can be suspected based on ECG findings. Autopsy study and clinical screening of family members are required to differentiate between Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia and chronic acquired myocarditis

Synthèse et évaluation biologique de molécules hétérobicycliques dans l'étude des interactions antagonistes entre Pseudomonas aeruginosa et Staphylococcus aureus chez les patients atteints de mucoviscidose

Cystic fibrosis (CF) is a genetic disease due to a gene mutation, that modifies the synthesis and the function of the CFTR protein (Cystic Fibrosis Trans-membrane conductance Regulator), a chloride channel found in different human organs. This modification induces ionic transport defaults. Particularly, in lungs, CF is responsible for an increase of the mucus viscosity. As a consequence, mucociliary clearance is decreased and the alveoli obstructed, leading to a suitable environment for pathogens development. In CF patient, differences exist between children and adults. Indeed, Staphylococcus aureus (Sa) is the major pathogen in young patients, but over the time Sa decreases in favor of Pseudomonas aeruginosa (Pa). Numerous studies have been conducted to understand the relationship between Pa-Sa. It could be explained by different mechanisms including the secretion in the environment of molecules from one microorganism and perceived by the another one. Understanding how the two bacteria interact and most of all, how Pa, in the respiratory tracts, takes over other bacteria, including Sa, is important to explain the physiopathology of lungs infections induced by Pa. We focused on the outcomes of these molecules, AQ, secreted by Pa.Thanks to previous works concerning the antagonist interactions between Pa-Sa, a hypothesis has been issued: molecules derivatives from 4-hydroxyquinoline are responsible of inhibitory activity of Pa onto Sa. The goals defined for this work consist : (i) in the synthesis of three classes of molecules (2-AQ-Cn, AQ-Cn, et 2-AQNO-Cn), (ii) in the biological evaluation of synthetized molecules, (iii) in a structure-activity relationship studyLa mucoviscidose (MV) est une maladie génétique rare, causée par la mutation génétique du gène CFTR. Ce gène code un transporteur des ions chlorures, appelé CFTR (Cystic Fibrosis Trans-membrane conductance Regulator) que l’on trouve dans divers organes du corps humain. Cette mutation entraine un défaut du transport ionique. La MV est responsable d’une augmentation de la viscosité du mucus, particulièrement dans les poumons. Cela engendre des conditions environnementales favorables à l’installation de pathogènes et à l’apparition d’infections respiratoires.Chez les patients MV il existe des différences entre les enfants et les adultes. En effet, Staphylococcus aureus (Sa) est le pathogène majeur chez les jeunes patients, tandis qu’avec le temps, il décroit au profit de Pseudomonas aeruginosa (Pa) chez l’adulte. Différentes études ont été réalisées pour comprendre les interactions Pa-Sa. Les mécanismes impliqués restent encore complexes. Cependant, ils pourraient être expliqués par la production et par la sécrétion, dans l’environnement pulmonaire, de molécules issues d’un micro-organisme et perçues par un second. Suite à des études préliminaires sur les interactions entre ces deux bactéries, une hypothèse a été émise : les molécules hétérobicycliques dérivées de la 4-hydroxyquinoléine sont responsables de l’activité inhibitrice de Pa sur Sa. Les objectifs que nous avons défini pour ce projet de thèse sont triple. Ils consistent : i) en la synthèse de composés appartenant à 3 familles dérivés de la 4-hydroxyquinoléine (2-AQ-Cn, AQ-Cn, et 2-AQNO-Cn), ii) en l’évaluation biologique des molécules synthétisées, iii) en une étude des relations structure-activit

Synthesis and biological evaluation of 4-hydroxyquinoline derivatives, study of antagonistic interactions between Pseudomonas aeruginosa and Staphylococcus aureus, in cystic fibrosis patients

La mucoviscidose (MV) est une maladie génétique rare, causée par la mutation génétique du gène CFTR. Ce gène code un transporteur des ions chlorures, appelé CFTR (Cystic Fibrosis Trans-membrane conductance Regulator) que l’on trouve dans divers organes du corps humain. Cette mutation entraine un défaut du transport ionique. La MV est responsable d’une augmentation de la viscosité du mucus, particulièrement dans les poumons. Cela engendre des conditions environnementales favorables à l’installation de pathogènes et à l’apparition d’infections respiratoires.Chez les patients MV il existe des différences entre les enfants et les adultes. En effet, Staphylococcus aureus (Sa) est le pathogène majeur chez les jeunes patients, tandis qu’avec le temps, il décroit au profit de Pseudomonas aeruginosa (Pa) chez l’adulte. Différentes études ont été réalisées pour comprendre les interactions Pa-Sa. Les mécanismes impliqués restent encore complexes. Cependant, ils pourraient être expliqués par la production et par la sécrétion, dans l’environnement pulmonaire, de molécules issues d’un micro-organisme et perçues par un second. Suite à des études préliminaires sur les interactions entre ces deux bactéries, une hypothèse a été émise : les molécules hétérobicycliques dérivées de la 4-hydroxyquinoléine sont responsables de l’activité inhibitrice de Pa sur Sa. Les objectifs que nous avons défini pour ce projet de thèse sont triple. Ils consistent : i) en la synthèse de composés appartenant à 3 familles dérivés de la 4-hydroxyquinoléine (2-AQ-Cn, AQ-Cn, et 2-AQNO-Cn), ii) en l’évaluation biologique des molécules synthétisées, iii) en une étude des relations structure-activitéCystic fibrosis (CF) is a genetic disease due to a gene mutation, that modifies the synthesis and the function of the CFTR protein (Cystic Fibrosis Trans-membrane conductance Regulator), a chloride channel found in different human organs. This modification induces ionic transport defaults. Particularly, in lungs, CF is responsible for an increase of the mucus viscosity. As a consequence, mucociliary clearance is decreased and the alveoli obstructed, leading to a suitable environment for pathogens development. In CF patient, differences exist between children and adults. Indeed, Staphylococcus aureus (Sa) is the major pathogen in young patients, but over the time Sa decreases in favor of Pseudomonas aeruginosa (Pa). Numerous studies have been conducted to understand the relationship between Pa-Sa. It could be explained by different mechanisms including the secretion in the environment of molecules from one microorganism and perceived by the another one. Understanding how the two bacteria interact and most of all, how Pa, in the respiratory tracts, takes over other bacteria, including Sa, is important to explain the physiopathology of lungs infections induced by Pa. We focused on the outcomes of these molecules, AQ, secreted by Pa.Thanks to previous works concerning the antagonist interactions between Pa-Sa, a hypothesis has been issued: molecules derivatives from 4-hydroxyquinoline are responsible of inhibitory activity of Pa onto Sa. The goals defined for this work consist : (i) in the synthesis of three classes of molecules (2-AQ-Cn, AQ-Cn, et 2-AQNO-Cn), (ii) in the biological evaluation of synthetized molecules, (iii) in a structure-activity relationship stud

Sterilisation in Dentistry: A Review of the Literature

In a small and medium-sized dental facility, the correct management of the sterilisation and presterilisation phases plays a fundamental role in good management of instruments and personnel, in order to ensure conditions that are more efficient with less down time. Nowadays, instrument sterilizers are increasingly efficient in achieving results, both in terms of time and size, and ensure that materials are sterile and ready to be stocked in a reasonable time. A literature search for articles related to revision work was performed using electronic databases such as PubMed, Scopus, and Google Scholar. The following keywords have been entered in the previously mentioned databases: sterilisation instruments; dental autoclave; precleaning; instruments disinfectants. The records obtained were screened by three reviewers, and only relevant articles were read full text. In addition, the timings of dental and sterilisation procedures were measured, and from these, suggestions are made in order to improve the efficiency of instrumentation management (facility used as study subject: University Dental Clinic, University of Foggia) as a function of the health-care interventions. We arrived at the conclusion that without doubt, sterilisation of instruments and products plays a fundamental role, but the efficiency of the sterilisation and presterilisation procedures cannot be separated from managing the personnel in charge by giving them specific and precise tasks

A Three-Step Process to Isolate Large Quantities of Bioactive Sesquiterpene Lactones from Cichorium intybus L. Roots and Semisynthesis of Chicory STLs Standards

International audienceSesquiterpene lactones (STLs) are a large group of terpenoids most commonly found in plants of the Asteraceae family, e.g., in chicory plants, displaying a wide range of interesting biological activities. However, further studies on the biological potential of chicory-derived STLs and analogues are challenging as only four of these molecules are commercially available (as analytical standards), and to date, there are no published or patented simple extraction–purification processes capable of large-scale STLs isolation. In this work, we describe a novel three-step large-scale extraction and purification method for the simultaneous purification of 11,13-dihydrolactucin (DHLc) and lactucin (Lc) starting from a chicory genotype rich in these STLs and the corresponding glucosyl and oxalyl conjugated forms. After a small-scale screening on 100 mg of freeze-dried chicory root powder, the best results were achieved with a 17 h water maceration at 30 °C. With these conditions, we managed to increase the content of DHLc and Lc, at the same time favoring the hydrolysis of their conjugated forms. On a larger scale, the extraction of 750 g of freeze-dried chicory root powder, followed by a liquid–liquid extraction step and a reversed-phase chromatography, allowed the recovery of 642.3 ± 76.3 mg of DHLc and 175.3 ± 32.9 mg of Lc. The two pure STLs were subsequently used in the context of semisynthesis to generate analogues for biological evaluation as antibacterial agents. In addition, other described chicory STLs that are not commercially available were also synthesized or extracted to serve as analytical standards for the study. In particular, lactucin-oxalate and 11,13-dihydrolactucin-oxalate were synthesized in two steps starting from Lc and DHLc, respectively. On the other hand, 11β,13-dihydrolactucin-glucoside was obtained after a MeOH/H2O (70/30) extraction, followed by a liquid–liquid extraction step and a reversed-phase chromatography. Together, this work will help facilitate the evaluation of the biological potential of chicory-derived STLs and their semisynthetic analogues