BackgroundThe stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates.MethodsThroughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC.ResultsWe demonstrated that SVF can be obtained with a consistent yield (about 185 × 103 cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (−11.6%). The relative abundance of ASC (CD34+CD45−CD31–) and endothelial precursors (CD34+CD45−CD31+) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 106 cells/ml. Cell viability was not significantly affected after a freezing period of 1 year.Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes.ConclusionsThis paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported.

Objective-Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury.FN containing Extra Domain A (EDA ϩ FN) is absent from normal plasma, but elevated plasma levels of EDA ϩ FN are found in several pathological conditions. We hypothesized that EDA ϩ FN plays a special role in thrombosis. Methods and Results-We used mouse strains constitutively including (EDA ϩ/ϩ ) or excluding (EDA Ϫ/Ϫ ) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model we found that EDA ϩ FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA ϩ/ϩ mice thrombi (Ͼ30 m) grew faster when compared with EDA WT/WT (6.6Ϯ0.2 minutes versus 8.3Ϯ0.6 minutes, PϽ0.05) and the mean vessel occlusion time was shorter (9.9Ϯ0.4 minutes versus 14.6Ϯ1.7 minutes, PϽ0.05). However, the presence of EDA ϩ FN affected neither single platelet adhesion to subendothelium nor thrombosis in veins. In addition, the mortality rate of EDA ϩ/ϩ mice after collagen/epinephrine infusion was twice that of EDA WT/WT or EDA Ϫ/Ϫ mice. Conclusions-Our findings reveal that EDAϩ FN has prothrombotic activity, and its presence in plasma may worsen pathological conditions in which this form is elevated. (Arterioscler Thromb Vasc Biol. 2008;28:296-301) Key Words: plasma fibronectin Ⅲ fibronectin splice variants isoforms Ⅲ arterial thrombosis Ⅲ intravital microscopy Ⅲ thromboembolism Ⅲ arterial and venous injury T he formation of a thrombus in an injured vessel wall is a complex process that involves multiple adhesion molecules and their respective receptors on the platelet surface. von Willebrand factor (vWF) and fibrinogen are considered the major ligands mediating platelet adhesion and aggregation. However, in an experimental model 73% of the injured vessels of mice lacking both fibrinogen and vWF still formed occlusive thrombi either at the site of injury or downstream, 1 suggesting that other major adhesive proteins, such as fibronectin (FN), might contribute to the process. Incorporation of FN into a growing thrombus was shown both in vitro 2 and in vivo. 3 Additionally, it was documented in vivo that the depletion or lower levels (50%) of plasma FN result in serious defects in arterial thrombosis. 3,4 FN is a dimeric multidomain glycoprotein playing an important role in adhesion, migration, growth and differentiation of cells. 5,6 FN generates protein diversity as a consequence of alternative processing of a single primary transcript at 3 sites: the Extra Domain B (EDB, EDII, or EIII-B), Extra Domain A (EDA, EDI, or EIII-A), and the Type III Homologies Connecting Segment (IIICS) ( Figure 1A). 7-9 Two major forms of FN exist: soluble plasma FN (pFN), which lacks both the EDA and EDB domains ( Figure 1A); and cellular FN (cFN), which is deposited as insoluble fibrils in the extracellular matrix (ECM) and contains these domains at variable proportions.FN is a ligand for many members of the integrin receptor family and binds to thrombosis-related protein...

© F e r r a t a S t o r t i F o u n d a t i o ndepletion and activates Orai1 (also known as calciumrelease-activated calcium-modulator, CRACM), the poreforming subunit of store-operated Ca 2+ channels. 19 The role of the additional STIM1 and Orai1 paralogs, i.e. STIM2 and Orai2-3, is far from being fully understood in naïve cell systems, albeit they may recapitulate SOCE when ectopically expressed. 19 Moreover, several evidences demonstrated that members of the canonical transient receptor potential (TRPC) family of cation channels may represent additional candidates for SOCE. 20 Agonistinduced elevation of intracellular Ca 2+ levels is essential for platelet activation. In this regard, STIM1 and Orai1 proteins have been shown to be key players in human platelet SOCE during aggregation, 21,22 whereas TRPC involvement has been questioned. 23,24 Importantly, it has been demonstrated that SOCE plays a major role in mediating adhesion and motility onto ECM components of different cell types, including hematopoietic stem cells.9,25 Thus we hypothesized that purinergic signaling and SOCE may be responsible for Mk interaction with the ECM environment and consequent regulation of platelet production. Our results demonstrate that ADP induces both intracellular Ca 2+ mobilization and extracellular Ca 2+ inflow in human Mks, which in turn support the cytoskeletal reorganization responsible for cellular adhesion and migration and final proplatelet formation on ECM components that promote such a dynamic process, such as fibrinogen and fibronectin. These findings provide the first evidence that Ca 2+ signaling is a fundamental regulator of human Mk functions. MethodsHuman cord blood was collected from the local blood bank following normal pregnancies and deliveries with informed consent of the parents, in accordance with the ethical committee of the IRCCS Policlinico San Matteo Foundation, Pavia, Italy, and the principles of the Declaration of Helsinki. CD34 + cells from cord blood samples were separated by immunomagnetic bead selection (Miltenyi Biotec, Bologna, Italy) and differentiated, as previously described. 4 At the end of cell culture, Mks were harvested and plated onto glass cover-slips previously coated with different ECM components in order to evaluate cell adhesion, migration and proplatelet formation. All images were acquired by Olympus BX51 microscope (Olympus, Deutschland GmbH, Hamburg, Germany). In some experiments, before being seeded, cells were pre-incubated with the following substances, at the indicated final concentrations: apyrase 1 U/mL, ADP 25 μM, 2-APB 20 µM, U-73122 10 μM, BTP-2 20 μM.We employed Ca 2+ imaging to investigate the expression and functionality of SOCE on the same extracellular matrix components. Specifically, Mks were loaded with 4 μM fura-2 acetoxymethyl ester (AM) or 5 mM FLUO-3 AM and observed using an upright epifluorescence Axiolab microscope (Carl Zeiss) equipped with a Zeiss X63 Achroplan objective or a laser-scanning confocal microscope (Nikon, Eclipse TE300).For all ...

BackgroundHepatitis C virus (HCV) has been consistently associated to non-Hodgkin lymphoma (NHL); conversely, few studies have evaluated a comprehensive serological panel of hepatitis B virus (HBV) in NHL etiology.MethodsWe conducted a case-control study in Italy in 1999–2014, enrolling 571 incident, histologically confirmed NHLs and 1004 cancer-free matched controls. Study subjects provided serum for HCV and HBV testing and for HCV RNA. Odds ratios (ORs) and corresponding 95 % confidence intervals (CIs) were estimated by logistic regression, adjusting for potential confounders.ResultsCirculating HCV RNA was detected in 63 (11.1 %) NHL cases and 35 (3.5 %) controls (OR = 3.51, 95 % CI: 2.25–5.47). Chronic HBV infection (i.e., positive to HBV surface antigen - HBsAg+) was found in 3.7 % of cases and 1.7 % of controls (OR = 1.95, 95 % CI: 1.00–3.81); a significantly elevated OR was observed for B-cell NHL (2.11, 95 % CI: 1.07–4.15). People with serological evidence of past HCV or HBV infection, vaccination against HBV, or detectable antibodies against HBV core antigen (anti-HBc+) alone were not at increased NHL risk.ConclusionsOur results support a role of chronic HCV infection in NHL in Italy and suggest an involvement of HBV infection. Associations were clearest for B-cell NHL and diffuse large B-cell lymphoma. Prevention and treatment of HCV and HBV infection may diminish NHL incidence, notably in areas with high prevalence of hepatitis viruses infection.

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BackgroundStandardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined.MethodsSupernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools.ResultsGrowth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)–CV); VEGF: 950–81.4; FGF-b: 27–74.6; PDGF-AA: 7883–28.8; PDGF-AB: 107834–32.5; PDGF-BB: 11142–48.4; Endostatin: 305034–16.2; Angiostatin: 197284–32.9; TGF-β1: 68382–53.7; IGF-I: 70876–38.3; EGF: 2411–30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential.DiscussionResults deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-017-1210-z) contains supplementary material, which is available to authorized users.

• The production of NO by platelets and its possible role are controversial.• We visualize NO formed by single platelets adhering to collagen under flow conditions and show that it depends on Ca 11 and modulates adhesion.Nitric oxide (NO) exerts vasodilatatory, antiplatelet, antioxidant, and antiproliferative effects. Endothelium-derived NO has been shown to be of crucial importance in cardiovascular protection, whereas evidence that NO is synthesized by platelets and regulates platelet function is still controversial. By using a sensitive and specific fluorescent probe, 4-amino-5-methylamino-29,79-difluorofluorescein diacetate (DAF-FM), we visualized NO production in individual platelets undergoing adhesion on a collagen substrate under flow conditions. NO production, monitored in real time, was dependent on the shear rates applied, increasing with the raising of the shear rates. Furthermore, NO production increased in the presence of L-arginine (nitric-oxide synthase [NOS] substrate), and it decreased in the presence of L-NG-monomethyl arginine (L-NMMA) (NOS inhibitor) but not of D-NG-monomethyl arginine (D-NMMA) (L-NMMA-inactive enantiomer). Platelet deposition, measured with mepacrine-labeled platelets, was inversely related to NO production. A correlation was evident between Ca 11 elevation and NO production, suggesting that platelet NO formation is triggered by intracytoplasmic Ca 11 elevation. Simultaneous measurement of NO and Ca 11 indicated that NO production in individual platelets is preceded by Ca 11 elevations, with a lag phase of 33 6 9.5 s. Our studies provide the first direct demonstration of platelet NO production triggered by the interaction with an activating surface under flow and suggest that intraplatelet Ca 11 elevation elicits the production of NO which, in turn, modulates thrombus size. (Blood. 2015;125(4):697-705)

Neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP)-9, and NGAL/MMP-9 complex have been evaluated as diagnostic markers of several cancers, but results for bladder cancer are scanty. We evaluated these proteins in urine and serum of 89 patients with histologically confirmed bladder cancer and 119 cancer-free controls from a case-control study. Urinary concentrations were standardized on creatinine level. The performance of these proteins as cancer biomarkers was evaluated through the receiver operating characteristic (ROC) analysis. Urinary level of NGAL, MMP-9, and NGAL/MMP-9 complex was higher in current smokers, whereas no impact of dietary habits was observed. After adjusting for tobacco smoking, urinary concentration of MMP-9 was independently associated with cancer invasiveness, grading, and histological subtype, with elevated concentrations among T2-T4 and non-papillary bladder cancers. Conversely, NGAL and NGAL/MMP-9 complex were significantly higher in non-papillary than in papillary subtype. The pattern was less clear in serum, but correlation between urinary and serum concentration was poor, especially for Ta/is-T1 tumors. The ROC analysis confirmed that MMP-9 was the best marker (area under the ROC curve (AUC) = 0.68). Performances were much greater for muscle-invasive bladder cancers (AUC = 0.90), with elevated negative predictive values (97 %). The present study suggests that NGAL/MMP-9 pathway is associated with an aggressive phenotype of bladder cancer. The elevated negative predictive value of MMP-9 and NGAL/MMP-9 complex makes them candidate markers of exclusion test for bladder cancer. These proteins may be integrated in the surveillance of bladder cancer, thus diminishing patients' discomfort and improving compliance.