S-CMC-Lys protective effects on human respiratory cells during oxidative stress.

The mucoactive drug S-carbocysteine lysine salt monohydrate (S-CMC-Lys) stimulates glutathione (GSH) efflux from respiratory cells. Since GSH is one of the most important redox regulatory mechanisms, the aim of this study was to evaluate the S-CMC-Lys effects on GSH efflux and intracellular concentration during an oxidative stress induced by the hydroxyl radical (xOH). Experiments were performed on cultured human respiratory WI-26VA4 cells by means of patch-clamp experiments in whole-cell configuration and of fluorimetric analyses at confocal microscope. xOH exposure induced an irreversible inhibition of the GSH and chloride currents that was prevented if the cells were incubated with S-CMC-Lys. In this instance, the currents were inhibited by the specific blocker CFTR(inh)-172. CFT1-C2 cells, which lack a functional CFTR channel, were not responsive to S-CMC-Lys, but the stimulatory effect of the drug was restored in LCFSN-infected CFT1 cells, functionally corrected to express CFTR. Fluorimetric measurements performed on the S-CMC-Lys-incubated cells revealed a significant increase of the GSH concentration that was completely hindered after oxidative stress and abolished by CFTR(inh)-172. The cellular content of reactive oxygen species was significantly lower in the S-CMC-Lys-treated cells either before or after xOH exposure. As a conclusion, S-CMC-Lys could exert a protective function during oxidative stress, therefore preventing or reducing the ROS-mediated inflammatory response

Coating of Titanium Substrates with ZrO2 and ZrO2-SiO2 Composites by Sol-Gel Synthesis for Biomedical Applications: Structural Characterization, Mechanical and Corrosive Behavior

The use of metallic materials as implants presents some major drawbacks, such as their harmful effects on the living organism, especially those induced by corrosion. To overcome this problem, the implant surface of titanium implants can be improved using a coating of bioactive and biocompatible materials. The aim of this work is the synthesis of SiO2/ZrO2 composites with different percentages of zirconia matrix (20, 33 and 50 wt.%), by the sol-gel method to coat commercial Grade 4 titanium disks using a dip coater. Attenuated total reflectance Fourier transform infrared (ATR/FTIR) spectroscopy was used to evaluate the interactions between the inorganic matrices. Furthermore, the mechanical properties and corrosive behavior of the SiO2/ZrO2 coatings were evaluated as a function of the ZrO2 content. The bioactive properties of the substrate coated with different composites were evaluated using simulated body fluid (SBF). The antibacterial activity was tested against gram-negative and gram-positive Escherichia coli and Enterococcus faecalis, respectively, to assess the release of toxic products from the different composites and to evaluate the possibility of using them in the biomedical field

Mitochondria-Ros Crosstalk in the Control of Cell Death and Aging

Reactive oxygen species (ROS) are highly reactive molecules, mainly generated inside mitochondria that can oxidize DNA, proteins, and lipids. At physiological levels, ROS function as “redox messengers” in intracellular signalling and regulation, whereas excess ROS induce cell death by promoting the intrinsic apoptotic pathway. Recent work has pointed to a further role of ROS in activation of autophagy and their importance in the regulation of aging. This review will focus on mitochondria as producers and targets of ROS and will summarize different proteins that modulate the redox state of the cell. Moreover, the involvement of ROS and mitochondria in different molecular pathways controlling lifespan will be reported, pointing out the role of ROS as a “balance of power,” directing the cell towards life or death

Reversible Monoacylglycerol Lipase Inhibitors: Discovery of a New Class of Benzylpiperidine Derivatives.

Monoacylglycerol lipase (MAGL) is the enzyme responsible for the metabolism of 2-arachidonoylglycerol in the brain and the hydrolysis of peripheral monoacylglycerols. Many studies demonstrated beneficial effects deriving from MAGL inhibition for neurodegenerative diseases, inflammatory pathologies, and cancer. MAGL expression is increased in invasive tumors, furnishing free fatty acids as pro-tumorigenic signals and for tumor cell growth. Here, a new class of benzylpiperidine-based MAGL inhibitors was synthesized, leading to the identification of 13, which showed potent reversible and selective MAGL inhibition. Associated with MAGL overexpression and the prognostic role in pancreatic cancer, derivative 13 showed antiproliferative activity and apoptosis induction, as well as the ability to reduce cell migration in primary pancreatic cancer cultures, and displayed a synergistic interaction with the chemotherapeutic drug gemcitabine. These results suggest that the class of benzylpiperidine-based MAGL inhibitors have potential as a new class of therapeutic agents and MAGL could play a role in pancreatic cancer

Design, synthesis, ADME and biological evaluation of benzylpiperidine and benzylpiperazine derivatives as novel reversible monoacylglycerol lipase (MAGL) inhibitors.

The degradation of the endocannabinoid 2-arachidonoylglycerol is mediated by the enzyme monoacylglycerol lipase (MAGL), thus generating arachidonic acid, the precursor of prostaglandins and other pro-inflammatory mediators. MAGL also contributes to the hydrolysis of monoacylglycerols into glycerol and fatty acids in peripheral body districts, which may act as pro-tumorigenic signals. For this reason, MAGL inhibitors have been considered as interesting therapeutic agents for their anti-nociceptive, anti-inflammatory, antioxidant and anti-cancer properties. So far, only a limited series of reversible MAGL inhibitors, which are devoid of side effects shown by irreversible inhibitors in animal models, have been reported. Here we optimized a class of benzylpiperidine and benzylpiperazine-based compounds for a reversible MAGL inhibition. The best MAGL inhibitors of this class, compounds 28 and 29, showed a very good inhibition potency, both on the isolated enzyme and in U937 cells, as confirmed by molecular modeling studies that predicted their binding mode into the MAGL active site. Both compounds are characterized by a high selectivity for MAGL versus other serine hydrolases including enzymes of the endocannabinoid system, as confirmed by ABPP experiments in mouse brain membranes. Moreover, very good properties concerning ADME parameters and low in vivo toxicity have been observed for both compounds

Detection of Merkel Cell Polyomavirus DNA in Serum Samples of Healthy Blood Donors

Merkel cell polyomavirus (MCPyV) has been detected in 80% of Merkel cell carcinomas (MCC). In the host, the MCPyV reservoir remains elusive. MCPyV DNA sequences were revealed in blood donor buffy coats. In this study, MCPyV DNA sequences were investigated in the sera (n = 190) of healthy blood donors. Two MCPyV DNA sequences, coding for the viral oncoprotein large T antigen (LT), were investigated using polymerase chain reaction (PCR) methods and DNA sequencing. Circulating MCPyV sequences were detected in sera with a prevalence of 2.6% (5/190), at low-DNA viral load, which is in the range of 1–4 and 1–5 copies/μl by real-time PCR and droplet digital PCR, respectively. DNA sequencing carried out in the five MCPyV-positive samples indicated that the two MCPyV LT sequences which were analyzed belong to the MKL-1 strain. Circulating MCPyV LT sequences are present in blood donor sera. MCPyV-positive samples from blood donors could represent a potential vehicle for MCPyV infection in receivers, whereas an increase in viral load may occur with multiple blood transfusions. In certain patient conditions, such as immune-depression/suppression, additional disease or old age, transfusion of MCPyV-positive samples could be an additional risk factor for MCC onset

Oxidative Stress in Cardiovascular Diseases and Obesity: Role of p66Shc and Protein Kinase C

Reactive oxygen species (ROS) are a byproduct of the normal metabolism of oxygen and have important roles in cell signalling and homeostasis. An imbalance between ROS production and the cellular antioxidant defence system leads to oxidative stress. Environmental factors and genetic interactions play key roles in oxidative stress mediated pathologies. In this paper, we focus on cardiovascular diseases and obesity, disorders strongly related to each other; in which oxidative stress plays a fundamental role. We provide evidence of the key role played by p66Shc protein and protein kinase C (PKC) in these pathologies by their intracellular regulation of redox balance and oxidative stress levels. Additionally, we discuss possible therapeutic strategies aimed at attenuating the oxidative damage in these diseases

CC Ligand 2 levels are increased in LPS-stimulated peripheral monocytes of patients with Non Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) shows a particular aggressive behaviour. Tumour associated macrophages (TAMs) play an important role in tumour growth and progression and CC ligand 2 (CCL2)/CCR2 axis is markedly involved in their recruitment in the tumour mass from the circulation. The aim of this study was to determine the plasma levels of CCL2 and the expression of CCR2 in the peripheral blood mononuclear cells (PBMCs) of 18 smokers with NSCLC, eight healthy smokers and nine non-smokers. Then, we investigated CCL2 levels in the supernatants of unstimulated and LPS-stimulated PBMC cultures of the same groups of patients. CCL2 levels in plasma and supernatants of PBMC cultures were determined by ELISA. CCR2 expression in PBMC cytospins was assessed by immunocytochemistry. CCL2 plasma levels and CCR2 expression by PBMCs were similar in patients with NSCLC, healthy smokers and non-smokers. In the supernatants of unstimulated PBMC cultures, CCL2 content was not different between the three groups of subjects. Supernatants of LPS-stimulated PBMCs of NSCLC patients showed a higher content of CCL2 as compared to supernatants of non-smokers (p<0.005). CCL2 content increased 28.5-fold vs baseline production in the group of NSCLC patients, 15-fold in healthy smokers and 13-fold in the group of non-smokers. In conclusion, after LPS stimulation, PBMCs of patients with NSCLC release higher levels of CCL2 as compared to those of non-smokers, supporting the hypothesis of a CCL2 involvement in NSCLC biology

Protocol for the long-term culture of human primary keratinocytes from the normal colorectal mucosa

Procedures for in vitro culturing of human primary keratinocytes from normal colon mucosa specimens have not been fully feasible, thus far. The protocol described herein allows primary keratinocytes from small tissue fragments of colorectal mucosa biopsies to grow in vitro. The procedure develops in three steps: (i) the enzymatic digestion of the tissue biopsy; (ii) the use of cloning rings to purify primary keratinocyte colonies, (iii) a defined keratinocyte medium to grow these cells in long-term culture. Our cultural method enables normal primary keratinocytes to be obtained by simple and rapid techniques. In our culture condition, primary keratinocytes express specific epithelial markers. Colorectal mucosa keratinocyte colonies require approximately two weeks to grow. Compared to previous approaches, our protocol provides a valuable model of study for human primary keratinocytes from normal colorectal mucosa both at the cellular and molecular levels. It is well known, that different mutations occurring during the multistep process of carcinogenesis in the normal colorectal mucosa, are strictly associated to the onset/progression of the colorectal carcinoma. On this ground, normal keratinocytes grown with our protocol, may represent an innovative tool to investigate the mechanisms that lead to colorectal carcinoma and other diseases. Our innovative procedure may allow to perform comparative investigations between normal and pathological colorectal cells