OSTEOBLAST-DERIVED EXTRACELLULAR VESICLES AND BONE

Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports investigated the role of EVs and their cargoes in cellular regulation and have tried to fine-tune their biotechnological use, but to date very little is known on their function in bone biology. To investigate the relevance of EV-mediated communication between bone cells, we isolated EVs from primary mouse osteoblasts and assessed membrane integrity, size and structure by transmission electron microscopy and fluorescence-activated cell sorting. EVs actively shuttled loaded fluorochromes to osteoblasts, monocytes and endothelial cells. Moreover, osteoblast EVs contained mRNAs shared with donor cells. Osteoblasts are known to regulate osteoclastogenesis, osteoclast survival and function by the pro-osteoclastic cytokine, Rankl. Osteoblast EVs were enriched in Rankl, which increased after PTH treatment. These EVs were biologically active, supporting osteoclast survival. EVs isolated from rankl-/- osteoblasts lost this pro-osteoclastic function, indicating its Rankl-dependence. They integrated ex-vivo into murine calvariae, and EV-shuttled fluorochromes were quickly uptaken by the bone upon in vivo EV systemic administration. Rankl-/- mice lack the osteoclast lineage and are negative for its specific marker TRAcP. Treatment of rankl-/- mice with wildtype osteoblast EVs induced the appearance of TRAcP-positive cells in an EV density-dependent manner. Finally, osteoblast EVs internalized and shuttled anti-osteoclast drugs (zoledronate and dasatinib), inhibiting osteoclast activity in vitro and in vivo. We conclude that osteoblast EVs are involved in intercellular communication between bone cells, contribute to the Rankl pro-osteoclastic effect and shuttle anti-osteoclast drugs, representing a potential means of targeted therapeutic delivery. This article is protected by copyright. All rights reserve

The future of stem cells in liver diseases.

Preliminary experience with clinical hepatocyte transplantation during the past decade has provided proof of concept that cell therapy can be effective for the treatment of some liver diseases. Recent progress in cell biology resulting in the isolation and characterization of hepatic stem cells and progenitor cells further increased the expectation for a new approach to the treatment of genetic and chronic liver disease. Several potential sources have been identified of hepatic stem/ progenitor cells exhibiting both differentiation towards the hepatic lineage in vitro and hepatic parenchymal repopulation with liver-specific metabolic activity in liver-injured animal models. However, a few of these results proved to be poorly reproducible in different laboratories, and it was recognized that some initial optimistic conclusions were drawn from incorrect interpretation of experimental data or from insufficient knowledge of the mechanisms involved in tissue regeneration. Moreover, only modest results have emerged so far from ongoing clinical experience involving the use of putative stem cells in liver disease. There is much need for a joined effort to concentrate the resources on a specific cell population, in order to better characterize its function, to assess its safety and Concise Revie

A simple method for the determination of lipid composition of human bile

The Entero-Test, a device for easy sampling of gastrointestinal contents, including bile, has been used for determination of biliary lipid composition. The device consists of a weighted gelatin capsule containing 140 cm of a highly absorbent nylon line. The capsule is swallowed while one end of the string is taped to the face. After 3.5 h, when the line has reached the duodenum, gallbladder contraction is stimulated by intramuscular administration of ceruletide. The line is pulled out, and the last 15 cm are eluted four times in methanol. Total bile acids (by 3 alpha-hydroxysteroid-dehydrogenase assay), individual bile acids (by high performance liquid chromatography), phospholipids (by assay of lipid-soluble phosphorus), and cholesterol (by gas-liquid chromatography) are determined in the eluate. Tests in vitro demonstrated no preferential binding and a good recovery of biliary lipids from the thread. Similar values of biliary cholesterol saturation were obtained by means of duodenal intubation and of the Entero-Test in a series of 12 subjects (r = 0.952). In 5 subjects, individual bile acids were also measured and were found to be similar with both techniques (r = 0.948). When the test was repeated over 3 days in a series of 7 subjects, biliary cholesterol saturation was found to be remarkably reproducible (CV = 7.6%). Thus, the Entero-Test is a convenient technique for the determination of biliary lipid composition, which can be particularly useful in longitudinal studies

Immunoregulatory Effects of Mesenchymal Stem Cell-Derived Extracellular Vesicles on T Lymphocytes.

The immunomodulatory activity of mesenchymal stem cells (MSCs) is largely mediated by paracrine factors. We have recently shown that the immunosuppressive effects of MSCs on B lymphocytes in peripheral blood mononuclear cell (PBMC) culture can be reproduced by extracellular vesicles (EVs) isolated from MSC culture supernatants. Here we investigated the effect of bone marrow-derived MSC-EVs on T cells on PBMC cultures stimulated with anti-CD3/CD28 beads. Stimulation increased the number of proliferating CD3+ cells as well as of regulatory T cells (Tregs). Coculture with MSCs inhibited the proliferation of CD3+ cells, with no significant changes in apoptosis. Addition of MSC-EVs to PBMCs did not affect proliferation of CD3+ cells, but induced the apoptosis of CD3+ cells and of the CD4+ subpopulation and increased the proliferation and the apoptosis of Tregs. Moreover, MSC-EV treatment increased the Treg/Teff ratio and the immunosuppressive cytokine IL-10 concentration in culture medium. The activity of indoleamine 2,3-dioxygenase (IDO), an established mediator of MSC immunosuppressive effects, was increased in supernatants of PBMCs cocultured with MSCs, but was not affected by the presence of MSC-EVs. MSC-EVs demonstrate immunomodulatory effects on T cells in vitro. However, these effects and the underlying mechanisms appear to be different from those exhibited by their cells of origin

High-dose fenoldopam reduces postoperative neutrophil gelatinase-associated lipocaline and cystatin C levels in pediatric cardiac surgery

21714857 2011 11 17 1466-609X 15 3 2011 Crit Care High-dose fenoldopam reduces postoperative neutrophil gelatinase-associated lipocaline and cystatin C levels in pediatric cardiac surgery. R160 The aim of the study was to evaluate the effects of high-dose fenoldopam, a selective dopamine-1 receptor, on renal function and organ perfusion during cardiopulmonary bypass (CPB) in infants with congenital heart disease (CHD).A prospective single-center randomized double-blind controlled trial was conducted in a pediatric cardiac surgery department. We randomized infants younger than 1 year with CHD and biventricular anatomy (with exclusion of isolated ventricular and atrial septal defect) to receive blindly a continuous infusion of fenoldopam at 1 \u3bcg/kg/min or placebo during CPB. Perioperative urinary and plasma levels of neutrophil gelatinase-associated lipocaline (NGAL), cystatin C (CysC), and creatinine were measured to assess renal injury after CPB.We enrolled 80 patients: 40 received fenoldopam (group F) during CPB, and 40 received placebo (group P). A significant increase of urinary NGAL and CysC levels from baseline to intensive care unit (ICU) admission followed by restoration of normal values after 12 hours was observed in both groups. However, urinary NGAL and CysC values were significantly reduced at the end of surgery and 12 hours after ICU admission (uNGAL only) in group F compared with group P (P = 0.025 and 0.039, respectively). Plasma NGAL and CysC tended to increase from baseline to ICU admission in both groups, but they were not significantly different between the two groups. No differences were observed on urinary and plasma creatinine levels and on urine output between the two groups. Acute kidney injury (AKI) incidence in the postoperative period, as indicated by pRIFLE classification (pediatric score indicating Risk, Injury, Failure, Loss of function, and End-stage kidney disease level of renal damage) was 50% in group F and 72% in group P (P = 0.08; odds ratio (OR), 0.38; 95% confidence interval (CI), 0.14 to 1.02). A significant reduction in diuretics (furosemide) and vasodilators (phentolamine) administration was observed in group F (P = 0.0085; OR, 0.22; 95% CI, 0.07 to 0.7).The treatment with high-dose fenoldopam during CPB in pediatric patients undergoing cardiac surgery for CHD with biventricular anatomy significantly decreased urinary levels of NGAL and CysC and reduced the use of diuretics and vasodilators during CPB.Clinical Trial.Gov NCT00982527. Pediatric Cardiac Anesthesia/Intensive Care Unit, Department of Pediatric Cardiology and Cardiac Surgery, Bambino Ges\uf9 Children's Hospital, Piazza S, Onofrio 4, 00165, Rome, Italy. [email protected] Ricci Zaccaria Z Luciano Rosa R Favia Isabella I Garisto Cristiana C Muraca Maurizio M Morelli Stefano S Di Chiara Luca L Cogo Paola P Picardo Sergio S eng ClinicalTrials.gov NCT00982527 Journal Article 20110629 England Crit Care 9801902 1364-8535 IM Crit Care. 2011;15(4):177 21861863 PMC3219034 2011413201151720116292011629201171602011716020117160epublishcc1029510.1186/cc1029521714857PMC321903

Engineered EVs for Oxidative Stress Protection

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.Fil: Tolomeo, Anna Maria. Università di Padova; ItaliaFil: Quarta, Santina. Università di Padova; ItaliaFil: Biasiolo, Alessandra. Università di Padova; ItaliaFil: Ruvoletto, Mariagrazia. Università di Padova; ItaliaFil: Pozzobon, Michela. Università di Padova; ItaliaFil: De Lazzari, Giada. Università di Padova; ItaliaFil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Turato, Cristian. Università di Padova; ItaliaFil: Arrigoni, Giorgio. Università di Padova; ItaliaFil: Pontisso, Patrizia. Università di Padova; ItaliaFil: Muraca, Maurizio. Università di Padova; Itali

Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1β, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.Fil: Tolomeo, Anna Maria. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Università di Padova; Italia. Consorzio per la Ricerca Sanitaria; ItaliaFil: Castagliuolo, Ignazio. Università di Padova; ItaliaFil: Piccoli, Martina. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Grassi, Michele. Università di Padova; ItaliaFil: Magarotto, Fabio. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Università di Padova; ItaliaFil: De Lazzari, Giada. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Consorzio per la Ricerca Sanitaria; Italia. Università di Padova; ItaliaFil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina. Consorzio per la Ricerca Sanitaria; Italia. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Caicci, Federico. Università di Padova; ItaliaFil: Franzin, Chiara. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Scarpa, Melania. Veneto Institute of Oncology; ItaliaFil: Macchi, Veronica. Università di Padova; ItaliaFil: De Caro, Raffaele. Università di Padova; Italia. Consorzio Per la Ricerca Sanitaria; ItaliaFil: Angriman, Imerio. Università di Padova; ItaliaFil: Viola, Antonella. Università di Padova; ItaliaFil: Porzionato, Andrea. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; ItaliaFil: Pozzobon, Michela. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Università di Padova; ItaliaFil: Muraca, Maurizio. Università di Padova; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Itali