Simone Guarnieri

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL) Sequentially Upregulates Nitric Oxide and Prostanoid Production in Primary Human Endothelial Cells

&NA; —Endothelial cells express tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptors, but the function of TRAIL in endothelial cells is not completely understood. We explored the role of TRAIL in regulation of key intracellular signal pathways in endothelial cells. The addition of TRAIL to primary human endothelial cells increased phosphorylation of endothelial nitric oxide synthase (eNOS), NOS activity, and NO synthesis. Moreover, TRAIL induced cell migration and cytoskeleton reorganization in an NO‐dependent manner. TRAIL did not activate the NF‐&kgr;B or COX‐2 pathways in endothelial cells. Instead, TRAIL increased prostanoid production (PGE2=PGI2>TXA2), which was preferentially inhibited by the COX‐1 inhibitor SC‐560. Because NO and prostanoids play a crucial role in the state of blood vessel vasodilatation and angiogenesis, our data suggest that TRAIL might play an important role in endothelial cell function. (Circ Res. 2003;92:732–740.)

Dysfunctional CFTR Alters the Bactericidal Activity of Human Macrophages against Pseudomonas aeruginosa

Chronic inflammation of the lung, as a consequence of persistent bacterial infections by several opportunistic pathogens represents the main cause of mortality and morbidity in cystic fibrosis (CF) patients. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes, additionally, mature macrophages from different tissues, although having a number of common activities, exhibit variation in some molecular and cellular functions. In order to highlight possible intrinsic macrophage defects due to CFTR dysfunction, we have focused our attention on in vitro differentiated macrophages from human peripheral blood monocytes. Here we report on the contribution of CFTR in the bactericidal activity against Pseudomonas aeruginosa of monocyte derived human macrophages. At first, by real time PCR, immunofluorescence and patch clamp recordings we demonstrated that CFTR is expressed and is mainly localized to surface plasma membranes of human monocyte derived macrophages (MDM) where it acts as a cAMP-dependent chloride channel. Next, we evaluated the bactericidal activity of P. aeruginosa infected macrophages from healthy donors and CF patients by antibiotic protection assays. Our results demonstrate that control and CF macrophages do not differ in the phagocytic activity when infected with P. aeruginosa. Rather, although a reduction of intracellular live bacteria was detected in both non-CF and CF cells, the percentage of surviving bacteria was significantly higher in CF cells. These findings further support the role of CFTR in the fundamental functions of innate immune cells including eradication of bacterial infections by macrophages.

Defective one- or two-electron reduction of the anticancer anthracycline epirubicin in human heart. Relative importance of vesicular sequestration and impaired efficiency of electron addition

One-electron quinone reduction and two-electron carbonyl reduction convert the anticancer anthracycline doxorubicin to reactive oxygen species (ROS) or a secondary alcohol metabolite that contributes to inducing a severe form of cardiotoxicity. The closely related analogue epirubicin induces less cardiotoxicity, but the determinants of its different behavior have not been elucidated. We developed a translational model of the human heart and characterized whether epirubicin exhibited a defective conversion to ROS and secondary alcohol metabolites. Small myocardial samples from cardiac surgery patients were reconstituted in plasma that contained clinically relevant concentrations of doxorubicin or epirubicin. In this model only doxorubicin formed ROS, as detected by fluorescent probes or aconitase inactivation. Experiments with cell-free systems and confocal laser scanning microscopy studies of H9c2 cardiomyocytes suggested that epirubicin could not form ROS because of its protonation-dependent sequestration in cytoplasmic acidic organelles and the consequent limited localization to mitochondrial one-electron quinone reductases. Accordingly, blocking the protonation-sequestration mechanism with the vacuolar H+-ATPase inhibitor bafilomycin A1 relocalized epirubicin to mitochondria and increased its conversion to ROS in human myocardial samples. Epirubicin also formed ∼60% less alcohol metabolites than doxorubicin, but this was caused primarily by its higher Km and lower Vmax values for two-electron carbonyl reduction by aldo/keto-reductases of human cardiac cytosol. Thus, vesicular sequestration and impaired efficiency of electron addition have separate roles in determining a defective bioactivation of epirubicin to ROS or secondary alcohol metabolites in the human heart. These results uncover the molecular determinants of the reduced cardiotoxicity of epirubicin and serve mechanism-based guidelines to improving antitumor therapies.

Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients.

BackgroundStenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.ResultsAll S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness.ConclusionsThe main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour

Aims:  This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development.

Calcium and Fos Involvement in Brain‐Derived Ca2+‐Binding Protein (S100)‐Dependent Apoptosis in Rat Phaeochromocytoma Cells

Brain‐derived calcium‐binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100‐induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c‐fos, c‐jun, bax, bcl‐x, p‐15, p‐21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L‐type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100‐derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L‐type Ca2+ channel conductance with consequent [Ca2+]i increase which up‐regulates, directly or indirectly, the expression of Fos.

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

Background/Aims. 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. Methods. Proliferative rates and neuritogenesis were tested by colorimetric assay and morphological analysis, respectively; reactive oxygen species (ROS) levels and intracellular Ca2+ variations monitored using single cell videomicroscopy. Results. 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 Ca2+ variations in undifferentiated cells, in which basal intracellular Ca2+ resulted increased after chronic exposure. In addition acute exposure affected cell response to a depolarizing agent, while basal membrane potential was not changed. Conclusion. Even if further studies remain necessary to identify the ROS/intracellular Ca2+cross-talking pathway activated by ELF-EMF exposure, we support the hypothesis that ROS and Ca2+ could be the cellular “primum movens” of the ELF-EMF induced effects on biological systems.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture

We have recently demonstrated that tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi‐quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. In our cellular model, TRAIL treatment not only increased NO levels but also caused a time‐dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co‐localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans‐cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL‐increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub‐cellular distribution is mediated by cytoskeleton and Golgi complex modifications. J. Cell. Biochem. 97: 782–794, 2006.

CD38 is constitutively expressed in the nucleus of human hematopoietic cells

CD38 is a type II glycoprotein that acts both as a bifunctional enzyme, responsible for the synthesis and hydrolysis of cyclic ADP‐ribose, and as a signal‐transducing surface receptor. Although CD38 was originally described as a plasma membrane molecule, several reports indicate that CD38 is expressed in the nucleus, even in cells known to be CD38 surface‐negative. In this study, firstly we investigated the presence of nuclear CD38 by immunofluorescence and confocal microscopy using a panel of hematopoietic cell lines that exhibit different levels of CD38 plasma membrane expression. Our second aim was to explore the relationship between the nuclear and plasma membrane forms of CD38 in human cell lines which represent discrete early maturation stages of the human lymphoid and myeloid compartments. Our results indicate that CD38 is constitutively present in the nucleus of cells belonging to distinct lineages. Furthermore, nuclear CD38 appears to be independent of the plasma membrane pool. The presence of nuclear CD38 during different stages of hematopoietic differentiation suggests that it may play a role in the control of nuclear Ca2+ homeostasis and NAD levels. J. Cell. Biochem. 105: 905–912, 2008.

Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.