Chrysotile, crocidolite, asbestiform erionite: mineralogical characterization and cytotoxic effects

UICC chrysotile, chrysotile from Val Malenco, erionite from Nevada and UICC crocidolite fibers were characterized through Transmission Electron Microscopy (TEM) with annexed Energy Dispersive Spectroscopy (EDS). The TEM study was performed forward on three levels for each single sample. The first observed aspect was the morphological and dimensional study: typical fibrous morphology was observed for all the analyzed samples. For each sample 100 fibers were investigated: were registered their dimension (length and diameter) and the L/d ratio were calculated in order to understand if the fibers population can entry in particular dimensional categories (e.g. WHO criteria, Stanton’s Hypothesis criteria or EPA dimensional limits). The second observed aspect was the chemical composition of the fibers. Also the chemistry match well with the expected for this minerals. In particular, both the chrysotile samples show the presence of aluminum and iron as substitute of tetrahedral and octahedral typical cations; the crocidolite bears an adding of calcium and the erionite has magnesium and iron cations normally unexpected in the general formula. At the latter investigation level, all the fibers showed a high degree of crystallinity in the diffraction patterns study, without evidence of natural amorphization (e.g. weathering). These characterized mineral fibres were administrated for 6, 12, 24 and 48h in human bronchial and mesothelial cells, at the concentration of 50µg/ml, to evaluate their cytotoxic effects; some biofunctional parameters at time points were evaluated: % number of alive, death and apoptotic cells; % number of cells with low, medium, high ROS content. These data confirm higher cytotoxic effects exerted by UICC crocidolite and UICC chrysotile, particularly evident since short times of contact (6, 12h). Our next purpose will be to characterize the same fibers extracted from cells after culture treatments

mRNAs and miRNAs profiling of Mesenchymal Stem Cells derived from amniotic fluid and skin

Mesenchymal Stem Cells (MSCs) may be isolated from different adult sources and even if the minimal criteria for defining MSCs have been reported, the scientific question about the potential distinctions among MSCs derived from different sources is still opened. In particular, it is debated if MSCs of different origin have the same grade of stemness or if the source affects their undifferentiated status. Here we report not only the isolation and the traditional characterization of MSCs derived from amniotic fluid (AF-MSCs) [1] and skin (S-MSCs) [2], but also a molecular characterization based on mRNAs and miRNAs profiling. Our results show that even if both AF- and S-MSCs are regulated by the same pathways (such as Wnt, MAPK and TGF-β), there is a fine and different control of them as suggested by altered levels of expression of some member of these pathways. In conclusion, it will be necessary to improve the knowledge about the role of each dysregulated miRs/gene because, actually, these differences may strengthen the question about the importance of tissue origin. This work was supported by grant FIRB-RBAP10MLK7_003 from Ministero dell’Istruzione, dell’Università e della Ricerca, Rome, Ital

In vitro biological effects of raw and thermally treated asbestos-containing materials

Asbestos cement, the main asbestos-containing material (ACM) manufactured in Italy in the past, is a health hazard whose elimination is a priority concern. Asbestos fibers can be transformed into potentially non-hazardous silicates by high-temperature treatment via complete solid-state transformation. In this study human A549 cells were directly exposed to raw cement asbestos (RCA), chrysotile and cement asbestos subjected to an industrial process at 1200 °C (HT-CA) and raw commercial grey cement (GC) for 24 and 48h, or treated with conditioned culture medium up to 96 h. In our previous studies we demonstrated that the final product of heat treatment of cement asbestos was considerably more inert and had lower cytotoxic potential than the original asbestos material. However, to better evaluate the risks of interactions with the materials, further in vitro investigations were performed concerning fiber-cell superficial interactions, immuno-hystochemical expression of cytochines p53, p53 homologue p73, TNF-related apoptosis- inducing ligand (TRAIL), and conditioned medium effects on cell viability. Data showed more severe cytotoxic damage by raw cement-asbestos compared to the heat treated materials and different expressions of cytochines that exert critical role in regulating the cell response to asbestos-induced DNA damage. These data should be taken in consideration for a safe recycling of thermal transformed asbestos materials

Attenuation of Listeria monocytogenes Virulence by Cannabis sativa L. Essential Oil

Anti-virulence strategies are being explored as a novel approach to combat pathogens. Such strategies include inhibition of surface adhesion, tissue invasion, toxin production, and/or interference with the gene regulation of other virulence traits. Listeria monocytogenes, the causative agent of listeriosis, is a facultative intracellular food pathogen characterized by a wide distribution in the environment. Its ability to persist within biofilms and to develop resistance to sanitizers is the cause of significant problems in food processing plants and of steep costs for the food industry. In humans, the treatment of listeriosis is hampered by the intracellular location of listeriae and the poor intracellular penetration of some antibiotics. Eleven L. monocytogenes isolates from patients who were diagnosed with invasive listeriosis in Italy in 2014–2016 were studied. This in vitro and in vivo study explored the antibacterial and anti-virulence properties of a steam-distilled essential oil of Cannabis sativa L., which is being intensively investigated for its high content in powerful bioactive phytochemicals. Susceptibility experiments demonstrated a moderate bactericidal activity of the essential oil (Minimum Bactericidal Concentration > 2048 μg/mL). Assessment of the effects of sublethal concentrations of the essential oil on L. monocytogenes virulence traits demonstrated a significant action on motility. Listeriae were non-motile after exposure to the essential oil. Light and scanning electron microscopy documented aggregates of listeriae with the flagella trapped inside the cluster. Real-time RT-PCR experiments showed downregulation of flagellar motility genes and of the regulatory gene prfA. The ability to form biofilm and to invade Caco-2 cells was also significantly reduced. Galleria mellonella larvae infected with L. monocytogenes grown in presence of sublethal concentrations of the essential oil showed much higher survival rates compared with controls, suggesting that the extract inhibited tissue invasion. Food contamination with L. monocytogenes is a major concern for the food industry, particularly for plants making ready-to-eat and processed food. The present work provides a baseline in the study of the anti-virulence properties of the C. sativa essential oil against L. monocytogenes. Further studies are needed to understand if it could be used as an alternative agent for the control of L. monocytogenes in food processing plants

43rd NATIONAL CONGRESS OF THE ITALIAN SOCIETY OF MICROBIOLOGY

Introduction: Capsaicin (8-methyl-N-vanillyl- 6-nonenamide) is the active component responsible for the fruit pungency of Capsicum plants, cultivated for food and also for medicinal uses since ancient times. Besides its multiple pharmacological and physiological properties (pain relief, cancer prevention, weight reduction, cardiovascular, and gastrointestinal benefits), capsaicin has recently received attention because of its antimicrobial activity and anti-virulence properties. The aim of the present study was to investigate the effects of capsaicin on Streptococcus pyogenes, the most common cause of acute bacterial pharyngotonsillitis. Materials and Methods: The erythromycin-resistant [erm(B)/cMLS], high cell-invasive, and strong biofilm producer S. pyogenes pharyngeal isolate SP1070 was used throughout the study. Capsaicin was purchased from Sigma-Aldrich and stored (10 mg/mL stock solution) in absolute ethanol at -20°C. The MIC and MBC were determined according to the CLSI guidelines. Survival in presence of capsaicin was studied by the live/dead assay. Biofilm formation was tested by a microtiter assay and quantified by measuring the absorbance at 690 nm. Cell experiments were performed using the human alveolar carcinoma A549 cell line. Results: The MIC and the MBC of capsaicin were both 128 μg/mL. In the live/dead assay, several red cells were detected as early as 15 min after incubation with capsaicin at MIC; all cells were red after 60 min of incubation. At capsaicin sub-MICs (1/2– 1/16 × MIC), a significant increase in biofilm production and in the number of streptococci adherent to A549 cells was observed; whereas a strong reduction in the number of intracellular bacteria was detected. Discussion and Conclusions: Our findings reveal that capsaicin has a dual effect on S. pyogenes. High-level capsaicin exerts a bactericidal effect, probably due to the disruption of the cell membrane, this result being in agreement with previous studies on Gram-positive and Gram-negative pathogens; while sub-lethal capsaicin modifies virulence properties in vitro, such as the ability to form biofilm and to adhere/invade epithelial cells. Capsaicin-induced effects on biofilm formation seem to be similar to those observed for a variety of antibiotics that at sub-lethal concentrations can act as agonists of bacterial biofilm production in vitro. Overall, capsaicin-induced effects on S. pyogenes deserve further studies