Circulating Extracellular Vesicles Impair Mesenchymal Stromal Cell Differentiation Favoring Adipogenic Rather than Osteogenic Differentiation in Adolescents with Obesity.

Excess body weight has been considered beneficial to bone health because of its anabolic effect on bone formation; however, this results in a poor quality bone structure. In this context, we evaluated the involvement of circulating extracellular vesicles in the impairment of the bone phenotype associated with obesity. Circulating extracellular vesicles were collected from the plasma of participants with normal weight, as well as overweight and obese participants, quantified by flow cytometry analysis and used to treat mesenchymal stromal cells and osteoblasts to assess their effect on cell differentiation and activity. Children with obesity had the highest amount of circulating extracellular vesicles compared to controls. The treatment of mesenchymal stromal cells with extracellular vesicles from obese participants led to an adipogenic differentiation in comparison to vesicles from controls. Mature osteoblasts treated with extracellular vesicles from obese participants showed a reduction in differentiation markers in comparison to controls. Children with obesity who regularly performed physical exercise had a lower circulating extracellular vesicle amount in comparison to those with a sedentary lifestyle. This pilot study demonstrates how the high amount of circulating extracellular vesicles in children with obesity affects the bone phenotype and that physical activity can partially rescue this phenotype.

Calibrated automated thrombogram values in infants with cardiac surgery before and after cardiopulmonary bypass.

INTRODUCTION: Impaired thrombin generation has been associated to increase bleeding after cardiac surgery with cardiopulmonary bypass (CPB), especially in children. The aim of this study was to evaluate standard coagulation assay, thrombin generation by calibrated automated thrombogram (CAT), thromboelastography (TEG) and procoagulant phospholipids (PPL) activity in infants undergoing cardiac surgery with CPB. MATERIALS AND METHODS: Prospective observational study performed in children aged <24months undergoing cardiac surgery with CPB. Exclusion criteria were preoperative coagulopathy or anticoagulant therapy. Coagulation was evaluated by standard coagulation assays (prothrombin time, activated partial thromboplastin time, fibrinogen level, platelet count), TEG, CAT and PPL at anaesthesia induction (T1) and after 12h (T2). Perioperative bleeding management was performed according to the institutional guidelines. RESULTS: Forty-nine children aged <24months were enrolled. At T1 ETP and peak height evaluated by CAT were significantly lower in infants aged <6months. Standard coagulation tests, TEG and PPL did not correlate with age. At T2 platelet count, plasmatic fibrinogen level, all TEG parameters, ETP and peak height by CAT were significantly impaired compared to baseline values (T1), despite allogeneic blood product transfusions. CONCLUSIONS: Thrombin generation is significantly impaired in children affected by congenital heart disease, compared to healthy children and adults. CAT parameters resulted age-dependent, and thrombin generation is lower in infants aged <6months. After cardiac surgery with CPB, a coaugulopathy, revealed by CAT, TEG, but not by PT and aPTT assays, is persistent 12h after surgery despite transfusions of blood products.

New Perspectives in Glioblastoma: Nanoparticles-based Approaches.

Glioblastoma multiforme represents one of the most aggressive tumor of central nervous system. Current therapy includes surgery, radiation and chemotherapy. These treatments are rarely curative and glioma are associated with a poor prognosis. Nanomedicine represents the most innovative branch of medicine since many studies demonstrated great advantage in the diagnosis and therapy of several diseases. In this review we will summarize the results obtained by the use of nanoparticles and extracellular vesicles in glioblastoma. A great interest is raising from these studies that underlined the efficacy and specificity of this treatment for glioma, reducing side-effects associated with conventional therapies.

Nanoparticles-Based Treatment for Bone Metastasis.

Bone is the principal site of metastasis for many carcinomas, including prostate. Once bone metastases are established, the chances of survival dramatically drop. Bone metastases place patients at increased risk of skeletal-related events, including pathologic fractures, bone pain and hypercalcemia. Indeed, skeletal metastases represent the prevalent cause of morbidity and mortality for many tumors. They are the result of interactions among tumour cells, bone marrow environment and bone cells (vicious cycle). In the last few years many efforts were undertaken to identify new therapeutic approaches for bone metastasis. Current therapies target the several players of bone vicious cycle. However many adverse effects are associated with these treatments. This review will focus on the new emerging sector of nanomedicine, that could be important to identify more specific and safe treatments for bone metastasis.

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.

Differential effects of extracellular vesicles secreted by mesenchymal stem cells from different sources on glioblastoma cells.

BACKGROUND: Malignant glial tumors, including glioblastoma multiforme, account for 15 - 20% of pediatric CNS malignancies. They are most resistant to therapy and are associated with a poor prognosis. OBJECTIVE: Given the ability of mesenchymal stem cells (MSCs) to affect glioma growth, we investigated the effects of extracellular vesicles (EVs) derived from MSCs on U87MG glioblastoma cells line. METHODS: EVs were isolated from culture media of MSCs from different sources, including bone marrow (BM), umbilical cord (UC) and adipose tissue (AT) and added to U87MG culture. The internalization and the effects of BM-, UC- and AT-MSC-EVs on proliferation and apoptosis of tumor cells were evaluated. RESULTS: Both confocal microscopy and FACS analysis showed internalization of EVs into tumor cells. BM- and UC-MSC-EVs decreased cell proliferation, while an opposite effect was observed with AT-MSC-EVs. Moreover, both BM- and UC-MSC-EVs induced apoptosis of glioblastoma cells, while AT-MSC-EVs had no effect. Loading UC-MSC-EVs with Vincristine further increased cytotoxicity when compared both to the free drug and to untreated EVs. CONCLUSIONS: Different effects of MSC-EVs on cancer cells were observed depending on their tissue of origin. Moreover, MSC-EVs can deliver antiblastic drugs to glioblastoma cells.

Recent advances in mesenchymal stem cell immunomodulation: the role of microvesicles.

Mesenchymal stem cells are the most widely used cell phenotype for therapeutic applications, the main reasons being their well-established abilities to promote regeneration of injured tissues and to modulate immune responses. Efficacy was reported in the treatment of several animal models of inflammatory and autoimmune diseases and, in clinical settings, for the management of disorders such as GVHD, systemic lupus erythematosus, multiple sclerosis, and inflammatory bowel disease. The effects of mesenchymal stem cells are believed to be largely mediated by paracrine signals, and several secreted molecules have been identified as contributors to the net biological effect. Recently, it has been recognized that bioactive molecules can be shuttled from cell to cell packed in microvesicles, tiny portions of cytoplasm surrounded by a membrane. Coding and noncoding RNAs are also carried in such microvesicles, transferring relevant biological activity to target cells. Several reports indicate that the regenerative effect of mesenchymal stem cells can be reproduced by microvesicles isolated from their culture medium. More recent evidence suggests that the immunomodulatory effects of mesenchymal stem cells are also at least partially mediated by secreted microvesicles. These findings allow better understanding of the mechanisms involved in cell-to-cell interaction and may have interesting implications for the development of novel therapeutic tools in place of the parent cells.

Insulin dynamics in young women with polycystic ovary syndrome and normal glucose tolerance across categories of body mass index.

BACKGROUND: Evidence favours insulin resistance and compensatory hyperinsulinemia as the predominant, perhaps primary, defects in polycystic ovary syndrome (PCOS). The aim of the present study was to evaluate insulin metabolism in young women with PCOS but normal glucose tolerance as compared with age, body mass index and insulin resistance-matched controls to answer the question whether women with PCOS hypersecrete insulin in comparison to appropriately insulin resistance-matched controls. RESEARCH DESIGN AND METHODS: Sixty-nine cases were divided according to their body mass index (BMI) in normal-weight (N = 29), overweight (N = 24) and obese patients (N = 16). Controls were 479 healthy women (age 16-49 y). Whole body Insulin Sensitivity (WBISI), fasting, and total insulin secretion were estimated following an oral glucose tolerance test (C-peptide deconvolution method). RESULTS: Across classes of BMI, PCOS patients had greater insulin resistance than matched controls (p<0.0001 for all the comparisons), but they showed higher fasting and total insulin secretion than their age, BMI and insulin resistance-matched peers (p<0.0001 for all the comparisons). CONCLUSION: Women with PCOS show higher insulin resistance but also larger insulin secretion to maintain normal glucose homeostasis than age-, BMI- and insulin resistance-matched controls.

Endocrine autoimmunity in Turner syndrome.

BACKGROUND: Turner syndrome is caused by numeric and structural abnormalities of the X chromosome. An increased frequency of autoimmunity as well as an elevated incidence of autoantibodies was observed in Turner patients. The aim of this study was to conduct a retrospective analysis of the incidence of autoimmunity in 66 Italian patients affected by Turner syndrome. METHODS: Sixty-six unselected and consecutive Italian Turner patients were recruited. The association between age, karyotype and the presence of clinical/pre-clinical autoimmune disorders and of autoantibodies was examined. RESULTS: Out of the 66 Turner patients, 26 had thyroid autoimmune disorders (39.4%), 14 patients had Hashimoto's thyroiditis with clinical or subclinical hypothyroidism (21.2%) and 12 patients had circulating anti-thyroid antibodies, echographic pattern of diffuse hypoechogenicity and normal thyroid hormone levels (18.2%). None were affected by Graves' disease. We analyzed the overall incidence of thyroid autoimmunity within the 3 different age groups 0-9.9, 10-19.9 and 20-29.9 years. No statistically significant difference was observed in the incidence of thyroid autoimmunity within the age-groups (χ2-test p > 0.05).Out of the 66 patients, 31 patients had the 45,X karyotype; within this first group 14 out of 31 patients were affected by autoimmune thyroid disease. A second group of 29 patients included 19 patients with mosaicism, 5 patients with deletions and 5 patients with ring chromosome; out of these 29 patients 7 were affected by autoimmune thyroid disease. A third group included 6 patients with X isochromosome; 5 out of 6 were affected by autoimmune thyroid disease. A statistically significant difference in the frequency of thyroid autoimmunity within the different karyotype groups was observed (χ2-test p = 0.0173).When comparing the X isochromosome group with the pooled group of other karyotypes, of note, the frequency of thyroid autoimmunity was statistically higher in the X isochromosome group (Fisher exact test p = 0.0315). CONCLUSIONS: Our data confirm a high frequency of thyroid autoimmunity in Italian Turner patients. Patients with X isochromosome are more prone to develop thyroid autoimmunity. Further, an early assay of autoantibodies and monitoring thyroid hormones is fundamental for detecting hypothyroidism earlier and start adequate replacement therapy.

The immunosuppressive effect of mesenchymal stromal cells on B lymphocytes is mediated by membrane vesicles.

The immunomodulatory properties of mesenchymal stromal cells are the subject of increasing interest and of widening clinical applications, but the reproducibility of their effects is controversial and the underlying mechanisms have not been fully clarified. We investigated the transfer of membrane vesicles, a recently recognized pathway of intercellular communication, as possible mediator of the interaction between mesenchymal stromal cells and B lymphocytes. Mesenchymal stromal cells exhibited a strong dose-dependent inhibition of B-cell proliferation and differentiation in a CpG-stimulated peripheral blood mononuclear cell coculture system. We observed that these effects could be fully reproduced by membrane vesicles isolated from mesenchymal stromal cell culture supernatants in a dose-dependent fashion. Next, we evaluated the localization of fluorescently labeled membrane vesicles within specific cell subtypes both by flow cytometry and by confocal microscopy analysis. Membrane vesicles were found to be associated with stimulated B lymphocytes, but not with other cell phenotypes (T lymphocytes, dendritic cells, natural killer cells), in peripheral blood mononuclear cell culture. These results suggest that membrane vesicles derived from mesenchymal stromal cells are the conveyors of the immunosuppressive effect on B lymphocytes. These particles should be further evaluated as immunosuppressive agents in place of the parent cells, with possible advantages in term of standardization, safety, and feasibility.

Impact of severe sepsis on serum and urinary biomarkers of acute kidney injury in critically ill children: an observational study.

BACKGROUND/AIMS: We hypothesized that sepsis could have an impact on the sensitivity of serum and urinary neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C (CysC) for acute kidney injury (AKI) diagnosis in critically ill children. METHODS: Serum NGAL (sNGAL) and urinary NGAL (uNGAL) and CysC were measured daily in the first 48 h from pediatric intensive care unit admission in 11 consecutive critically ill children with severe sepsis; a single measurement was made in a population of 10 healthy controls undergoing minor ambulatory surgery to exclude possible biases in the laboratory methods. RESULTS: uNGAL, serum CysC (sCysC), and urinary CysC (uCysC) levels were significantly increased in patients with septic AKI compared with septic patients without AKI, while sNGAL levels were not significantly different between septic patients with and without AKI. Median serum creatinine levels did not show significant differences between AKI and non-AKI patients. CONCLUSIONS: uNGAL, sCysC and uCysC were not altered by sepsis and were good predictors of AKI. In a septic state, sNGAL alone did not discriminate patients with AKI from those without AKI.

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

INTRODUCTION: 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). METHODS: 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 µg/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. RESULTS: 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). CONCLUSIONS: 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. TRIAL REGISTRATION: Clinical Trial.Gov NCT00982527.