Effects of acute and chronic low frequency electromagnetic field exposure on PC12 cells during neuronal differentiation.

The purpose of this study was to provide information about the in vitro neuritogenesis during cell exposure to extremely low frequency electromagnetic fields (ELF-EMFs) of different intensities and durations using pheochromocytoma-derived cell line (PC12 cells) as neuronal model.Proliferative rates and neuritogenesis were tested by colorimetric assay and morphological analysis, respectively; reactive oxygen species (ROS) levels and intracellular Ca(2+) variations monitored using single cell videomicroscopy.The long-lasting ELF-EMF exposure (0.1-1.0 mT) did not appear to significantly affect the biological response (proliferation and neuritogenesis). However, during the acute ELF-EMF exposure (30 min), in undifferentiated PC12 cells, there were increased ROS levels and decreased catalase activity, that, conversely, resulted increased after chronic exposure (7 days) at 1.0 mT. Acute exposure (0.1-1.0 mT) affected the spontaneous intracellular Ca(2+) variations in undifferentiated cells, in which basal intracellular Ca(2+) resulted increased after chronic exposure. In addition acute exposure affected cell response to a depolarizing agent, while basal membrane potential was not changed.Even if further studies remain necessary to identify the ROS/intracellular Ca(2+)cross-talking pathway activated by ELF-EMF exposure, we support the hypothesis that ROS and Ca(2+) could be the cellular "primum movens" of the ELF-EMF induced effects on biological systems

hAFSC expressing a specific panel of stem cell markers give rise to fully differentiate cardiomyocytes

Human amniotic fluid-derived stem cells (hAFSC) represent a novel class of multipotent stem cells sharing characteristics of both embryonic and adult stem cells. In fact, hAFSC proliferate rapidly, are able to differentiate into cells of all the three embryonic germ layers, but do not form teratoma. It has been already reported that hAFSC have a cardiac potential, but a high variability between hAFSC donors in differentiation efficiency has been described. Aim of this study was to phenotypically identify the hAFSC able to differentiate into mature cardiomyocytes. hAFSCs from 10 different donors were characterized for the immunophenotypic expression of stemness markers and then cultured in differentiatve conditions. hAFSC differed for both stemness markers expression and for differentiation efficiency. Only the hAFSC expressing specific stem cell antigens were able to differentiate into a homogeneous population of cells that highly express cardiac cytoskeletal proteins and the structural and functional sarcoplasmatic reticulum proteins. Our results demonstrate that only hAFSC showing a specific stem cell pattern phenotype can fully differentiate into myocytes giving rise to a homogenous population characterized by cardiac-specific molecular, structural, and functional properties

Adhesion of human gingival fibroblasts/Streptococcus mitis co-culture on the nanocomposite system Chitlac-nAg

10noComposite materials are increasingly used as dental restoration. In the field of biomaterials, infections remain the main reason of dental devices failure. Silver, in the form of nanoparticles (AgNPs), ions and salt, well known for its antimicrobial properties, is used in several medical applications in order to avoid bacterial infection. To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized AgNPs with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a coculture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release raised up to 20 %. Moreover the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces with the co-culture system even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKCα translocated into nuclei. These data confirm that Chitlac-nAg have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.openopenCataldi, Amelia; Gallorini, Marialucia; Di Giulio, Mara; Guarnieri, Simone; Mariggiò, Maria Addolorata; Traini, Tonino; Di Pietro, Roberta; Cellini, Luigina; Marsich, Eleonora; Sancilio, SilviaCataldi, Amelia; Gallorini, Marialucia; Di Giulio, Mara; Guarnieri, Simone; Mariggiò, Maria Addolorata; Traini, Tonino; Di Pietro, Roberta; Cellini, Luigina; Marsich, Eleonora; Sancilio, Silvi

Human Cardiopoietic Amniotic Fluid cell population: characterization and terminal differentiation

Rationale. Human amniotic fluid-derived (hAF) stem cells are considered a novel class of multipotent stem cells, sharing characteristics of both embryonic and adult stem cells. In fact, they proliferate rapidly, are able to differentiate into cells of all the embryonic germ layers, but do not form teratoma. It has been already reported that the embryoid bodies (EBs) obtained from hAFs have a cardiac potential, but it has not been described a functional terminal differentiation in cardiomyocytes (CMs) yet. Objective. Aim of this study was to foster the cardiomyogenic potential of hAFs in order to obtain a cellular population with morphological and functional features of CMs. Methods and Results. AFCs were exposed sequentially to inducing factors (Ascorbic Acid, 5-Azacytidine, BMP4, ActivinA, VEGF) up to 15 days and differentiation was monitored. Only the hAF samples expressing the multipotency markers SSEA4, OCT4 and CD90 (CardiopoieticAF) responded to the differentiation process loosing their stemness and increasing the cardiac nuclear factors NKX2.5 and GATA4. After the differentiation cells expressed high levels of the sarcomeric proteins (cTnT, αMHC and αSA), the gap junction marker Connexin43 and both atrial and ventricular markers; moreover, up to 90% of the cells was positive for CACNA1C and SERCA2a, cardiac calcium pumps involved in the excitation/contraction coupling, and about 30% of the CardiopoieticAF-derived cells presented spontaneous intracellular Ca2+ waves and Ca2+ fluctuation in response to caffeine or adrenergic stimulation. Some spontaneous beating foci were also observed. Conclusion. Our results demonstrate that CardiopoietichAFs can fully differentiate into a homogenous population of CM-like cells, characterized by cardiac-specific molecular, structural, and functional properties. Thus, CardiopoietichAFs can hold great promise for the development of in vitro models of cardiac genetic disorders, for drug discovery and testing, and for the emerging field of cardiovascular regenerative medicine

Impact of Single Hemodialysis Treatment on immune Cell Subpopulations

: Hemodialysis (HD) is known to trigger a chronic inflammatory status, affecting the innate and acquired immune response. This study was aimed at a comparative analysis of immune cell subsets, proliferation, and apoptosis in subjects receiving chronic HD treatment with respect to a healthy control. Regardless of the dialysis filter used, we observed a reshaping of the acquired immune component both with respect to healthy patients and between the various sessions of dialysis treatment, with an impairment of CD3 cells, along with an increase in CD4 and CD8 cell populations producing pro-inflammatory factors such as IL-17 and IFN-gamma. The population of B cells, monocytes and NK cells were not impaired by the dialysis procedure. These results confirmed the high impact of the HD treatment on the patient's immune system, underlying the imbalance of T cell counterparts

Is expression of p120ctn in oral squamous cell carcinomas a prognostic factor?

Objectives p120ctn is a component of the catenin family. To date, there have only been two studies examining expression levels of p120ctn in oral squamous cell carcinoma (OSCC). Materials and methods Paraffined specimens of 113 OSCCs and 12 of normal mucosa were examined by immunohistochemistry. Frozen samples of 20 OSCCs and 5 of normal mucosa were examined by Western blot (WB). Results were correlated with clinicopathological parameters. Five cell lines were examined by immunofluorescence, immunocytochemistry, and WB to show immunoreactivity and cellular localization of p120ctn. Results Altered p120ctn expression was observed in 109/113 cases of OSCC. Heterogenous cytoplasmic/nuclear expression was associated with loss of membranous distribution (88/113 cases). Complete loss of expression was noted in 21/113 cases. Increased cytoplasmic expression was evident in all positive cases, without significant correlation among p120ctn staining/pattern and grading/stage. Reduction/absence of p120ctn expression was related to poor prognosis ( P Conclusion p120ctn delocalization/loss of expression could be an independent prognostic marker in OSCC

Local allergic rhinitis: entopy or spontaneous response?

Background The existence of a local allergic rhintis was proposed on the basis of the detection of nasal IgE in the absence of a systemic sensitization. Nevertheless, the significance of this phenomenon remains still unclear.We assessed the presence of mucosal nasal IgE in patients with ascertained allergic rhinitis, nonallergic rhinitis with inflammation and in healthy controls.Methods Consecutive patients with a well ascertained diagnosis (clinical history, skin prick test, specific IgE assay, nasal endoscopy, nasal cytology) underwent an immunoenzymatic measurement of specific IgE to grass, cypress, parietaria and olive in nasal scrapings.Results Fifteen patients with allergic rhinitis, 12 with non allergic rhinitis and 14 healthy subjects were studied. The patients with allergic and nonallergic rhinitis had higher nasal symptoms as compared to control subjects. Systemic sensitizatition (assessed by skin test and CAP-RAST) was obviously more frequent in allergic rhinitis, than in the other two groups. Allergen-specific nasal IgE could be detected in all groups (86,7, 33,3, and 50 % positive, respectively), even more frequently in the control group than in nonallergic rhinitis patients. No difference among allergens was identified. Out of the 26 non-allergic patients (non allergic rhinitis + controls) nasal IgE were positive in 11(42 %).Discussion According to the results, the presence of nasal IgE against allergens seems to be a non-specific phenomenon, since they can be detected also in non allergic rhinitis and in healthy subjects.Conclusion It can be hypothesized that the nasal IgE production represents a form of spontaneous immune response. Keywords: Allergic rhinitis, Nonallergic rhinitis, Sensitization, Nasal IgE, Local allergic rhinitis, Entop

Analytical Assessment of the Vela Diagnostics NGS Assay for HIV Genotyping and Resistance Testing: The Apulian Experience

Drug-resistance monitoring is one of the hardest challenges in HIV management. Next-generation sequencing (NGS) technologies speed up the detection of drug resistance, allowing the adjustment of antiretroviral therapy and enhancing the quality of life of people living with HIV. Recently, the NGS Sentosa® SQ HIV Genotyping Assay (Vela Diagnostics) received approval for in vitro diagnostics use. This work is the first Italian evaluation of the performance of the Vela Diagnostics NGS platform, assessed with 420 HIV-1 clinical samples. A comparison with Sanger sequencing performance is also reported, highlighting the advantages and disadvantages of the Sentosa® NGS assay. The precision of the technology was studied with reference specimens, while intra- and inter-assay reproducibility were evaluated for selected clinical samples. Vela Diagnostics’ NGS assay reached an 87% success rate through 30 runs of analysis in a real-world clinical context. The concordance with Sanger sequencing outcomes was equal to 97.2%. Several detected mismatches were due to NGS’s superior sensitivity to low-frequency variants. A high accuracy was observed in testing reference samples. Repeatability and reproducibility assays highlighted the good performance of the NGS platform. Beyond a few technical issues that call for further optimization, the key improvement will be a better balance between costs and processing speed. Once these issues have been solved, the Sentosa® SQ HIV Genotyping Assay will be the way forward for HIV resistance testing

Calcium sensing receptor expression in ovine amniotic fluid mesenchymal stem cells and the potential role of R-568 during osteogenic differentiation.

Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine