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71.
Elena De Falco Gaia Scafetta Chiara Napoletano Rosa Puca Enzo Maria Vingolo Giuseppe Ragona Olga Iorio Giacomo Frati 《Cell and tissue banking》2013,14(2):277-287
Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm2 undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes. 相似文献
72.
Francesca Borgo Aristodemo Carpen Chiara Ferrario Stefania Iametti Maria Grazia Fortina 《Journal of industrial microbiology & biotechnology》2013,40(5):489-494
Through the analysis of the recently available genome shotgun sequence of Enterococcus italicus DSM 15952T type strain (Accession PRJNA61487, ID 61487), we found the presence of a gene encoding a bifunctional enzyme, termed γ-GCS-GS or GshF, involved in glutathione production and not influenced by feedback inhibition. The gshF gene exhibited high nucleotide and amino acid sequence similarity to other reported sequences from the Enterococcus genus and was constitutively expressed both in osmotic shock or in common cultural conditions. Several experimental studies concerning the culture medium, physiological stress, cell extract obtainment, and scaling-up showed that in selected conditions E. italicus was able to accumulate up to 250 μM of intracellular glutathione, which represented the main thiol group present into the cells. This is the first report regarding the production of glutathione by E. italicus, a species that could be used as a safe adjunct culture for glutathione-enriched dairy foods. 相似文献
73.
Martina Bernardi Elena Albiero Alberta Alghisi Katia Chieregato Chiara Lievore Domenico Madeo Francesco Rodeghiero Giuseppe Astori 《Cytotherapy》2013,15(8):920-929
Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS. 相似文献
74.
Anna Licata Salvatore Corrao Salvatore Petta Chiara Genco Mauro Cardillo Vincenza Calvaruso Giuseppe Cabibbo Fatima Massenti Calogero Cammà Giuseppe Licata Antonio Craxì 《PloS one》2013,8(8)
Background and Aims
Plasma levels of NT-pro-BNP, a natriuretic peptide precursor, are raised in the presence of fluid retention of cardiac origin and can be used as markers of cardiac dysfunction. Recent studies showed high levels of NT pro BNP in patients with cirrhosis. We assessed NT pro-BNP and other parameters of cardiac dysfunction in patients with cirrhosis, with or without ascites, in order to determine whether the behaviour of NT pro BNP is linked to the stage of liver disease or to secondary cardiac dysfunction.Methods
Fifty eight consecutive hospitalized patients mostly with viral or NAFLD-related cirrhosis were studied. All underwent abdominal ultrasound and upper GI endoscopy. Cardiac morpho-functional changes were evaluated by echocardiography and NT-pro-BNP plasma levels determined upon admission. Twenty-eight hypertensive patients, without evidence of liver disease served as controls.Results
Fifty eight cirrhotic patients (72% men) with a median age of 62 years (11% with mild arterial hypertension and 31% with type 2 diabetes) had a normal renal function (mean creatinine 0.9 mg/dl, range 0.7–1.06). As compared to controls, cirrhotic patients had higher NT pro-BNP plasma levels (365.2±365.2 vs 70.8±70.6 pg/ml; p<0.001). Left atrial volume (LAV) (61.8±26.3 vs 43.5±14.1 ml; p = 0.001), and left ventricular ejection fraction (62.7±6.9 vs. 65.5±4%,; p = 0.05) were also altered in cirrhotic patients that in controls. Patients with F2-F3 oesophageal varices as compared to F0/F1, showed higher e'' velocity (0.91±0.23 vs 0.66±0.19 m/s, p<0.001), and accordingly a higher E/A ratio (1.21±0.46 vs 0.89±0.33 m/s., p = 0.006).Conclusion
NT-pro-BNP plasma levels are increased proportionally to the stage of chronic liver disease. Advanced cirrhosis and high NT-pro-BNP levels are significantly associated to increased LAV and to signs of cardiac diastolic dysfunction. NT pro-BNP levels could hence be an useful prognostic indicators of early decompensation of cirrhosis. 相似文献75.
Tullio Palmerini Luciana Tomasi Chiara Barozzi Diego Della Riva Andrea Mariani Nevio Taglieri Ornella Leone Claudio Ceccarelli Stefano De Servi Angelo Branzi Philippe Genereux Gregg W. Stone Jasimuddin Ahamed 《PloS one》2013,8(12)
Introduction
Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention.Methods
Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients.Results
Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p<0.001). Tissue factor was also identified by immunoprecipitation in 10 patients, with significant variability among band intensities.Conclusions
Active tissue factor is present in coronary artery thrombi of patients with ST-segment elevation acute myocardial infarction, suggesting that it contributes to activate the coagulation cascade ensuing in coronary thrombosis. 相似文献76.
Inmaculada Banon-Rodriguez Julia Saez de Guinoa Alejandra Bernardini Chiara Ragazzini Estefania Fernandez Yolanda R. Carrasco Gareth E. Jones Francisco Wandosell Ines Maria Anton 《PloS one》2013,8(8)
The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility. 相似文献
77.
Chiara Donfrancesco Simonetta Palleschi Luigi Palmieri Barbara Rossi Cinzia Lo Noce Fabio Pannozzo Belinda Spoto Giovanni Tripepi Carmine Zoccali Simona Giampaoli 《PloS one》2013,8(10)
Background
Chronic kidney disease (CKD) independently increases the risk of death and cardiovascular disease (CVD) in the general population. However, the relationship between estimated glomerular filtration rate (eGFR) and CVD/death risk in a general population at low risk of CVD has not been explored so far.Design
Baseline and longitudinal data of 1465 men and 1459 women aged 35-74 years participating to the MATISS study, an Italian general population cohort, were used to evaluate the role of eGFR in the prediction of all-cause mortality and incident CVD.Methods
Bio-bank stored sera were used to evaluate eGFR at baseline. Serum creatinine was measured on thawed samples by means of an IDMS-calibrated enzymatic method. eGFR was calculated by the CKD-EPI formula.Results
At baseline, less than 2% of enrolled persons had eGFR < 60 mL/min/1.73m2 and more than 70% had a 10-year cardiovascular risk score < 10%. In people 60 or more years old, the first and the last eGFR quintiles (<90 and ≥109 mL/min/1.73m2, respectively) were associated to an increased risk for both all-cause mortality (hazard ratio 1.6, 95% confidence interval 1.2-2.1 and 4.3, 1.6-11.7, respectively) and incident CVD (1.6, 1.0-2.4 and 7.0, 2.2-22.9, respectively), even if adjusted for classical risk factors.Conclusions
These findings strongly suggest that in an elderly, general population at low risk of CVD and low prevalence of reduced renal filtration, even a modest eGFR reduction is related to all-cause mortality and CVD incidence, underlying the potential benefit to this population of considering eGFR for their risk prediction. 相似文献78.
Thorsten Klampfl Jelena D. Milosevic Ana Puda Andreas Sch?negger Klaudia Bagienski Tiina Berg Ashot S. Harutyunyan Bettina Gisslinger Elisa Rumi Luca Malcovati Daniela Pietra Chiara Elena Matteo Giovanni Della Porta Lisa Pieri Paola Guglielmelli Christoph Bock Michael Doubek Dana Dvorakova Nada Suvajdzic Dragica Tomin Natasa Tosic Zdenek Racil Michael Steurer Sonja Pavlovic Alessandro M. Vannucchi Mario Cazzola Heinz Gisslinger Robert Kralovics 《PloS one》2013,8(10)
Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized. 相似文献
79.
Chiara Ciaccio Alessandra Pesce Grazia R. Tundo Lesley Tilleman Laura Bertolacci Sylvia Dewilde Luc Moens Paolo Ascenzi Martino Bolognesi Marco Nardini Massimo Coletta 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(9):1813-1823
Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. 相似文献
80.
Francesca Boggiano Claudio Ciofi Luigi Boitani Angela Formia Lorenza Grottoli Chiara Natali Paolo Ciucci 《Mammalian Biology》2013,78(5):374-378
Southern European wolves suffered from reiterated population declines during glacial periods and historically due to human persecution. Differently from other European wolf populations, a single mitochondrial DNA (mtDNA) control region haplotype (W14) has been so far described in the Italian wolves, although no intensive genetic sampling has ever been conducted in historical source populations from central and southern Italy. Using non-invasive genetic techniques, we report the occurrence of an unexpected mtDNA haplotype (W16) in the wolf population of the Abruzzo, Lazio and Molise National Park (PNALM), central Italy. This haplotype, detected in three out of 90 faecal samples from the PNALM, was previously reported in wolves from the North Carpathians, Slovakia and the Balkans only. Microsatellite analysis and molecular sex determination confirmed that the W16 samples belonged to three distinct wolves. Although alternative explanations can be formulated for the origin of this mtDNA haplotype in the otherwise monomorphic Italian wolf population, assignment procedures indicated the likely admixed ancestry of one W16 sample with East European wolves. Anthropogenic introgression with dogs has been detected in the Italian wolf population using nuclear DNA microsatellites, but no population-wide genetic survey had previously reported a mtDNA control region variant in Italian wolves. Our findings strongly suggest that, in addition to wolf × dog hybridization, captive-released wolves or wolf × dog hybrids may successfully interbreed with wolves in the wild, and that human-mediated introgression may occur even in well established protected areas. 相似文献