Interessante. 19 ago 2009 - Metti una sera prima di cena, ad Agliano Terme, nido di Danilo Sacco. In this study, we addressed the question whether MM-released exosomes detected by Id-peptides could allow a more efficient monitoring of tumor growth compared to the standard paraprotein assay. Pan BT, Teng K, Wu C, Adam M, Johnstone RM. The Igs secreted by 5T33MM cells were purified from cell supernatant using Protein G affinity chromatography, and used as bait to isolate phage ligands from the C7C phage-displayed peptide library fused to the M13 minor coat protein. The SEM analysis was performed at 20, 000 x magnification (SEM FEI Novalab 600). 2010;116(2):226–38. Cultured 5T33MM, A20 and IM9 cells as well as primary B cells from MM patient and healthy donor were washed and suspended at the density of 106 cells/ml in FACs Flow Buffer [BD Biosciences]. Situazione Sentimentale Sposato : L'orientamento sessuale Etero: Attuale Moglie di Danilo: Clarice Sales: ... Un sacco di celebrità non sa più cosa fare con i propri soldi. Théry C, Amigorena S, Raposo G. Clayton A. Curr Protoc Cell Biol. Flow cytometry analysis of purified exosomes derived from different cell sources (DOC 570 kb). Il 15-7-1991 Danilo (soprannome: Danilo) è nato a Bicas, Brazil. Dynamic light scattering and zeta potential determinations were performed with a Nano ZS 90 [Malvern Instruments], allowing the analysis of particles within the range of 1 nm up to 3 μm. Mol Cancer 16, 159 (2017). The 5T33MM cell culture supernatant was filtered (0.22 μm) and the exosomes were purified using the ExoQuick-TC™. Google Scholar. The insert peptide sequence from selected phage, the percentage of clonal identity, and KD values of identified synthetic Id-peptides are shown in Table 1. CAS  “Oh,” Danilo said, looking down at his cards, seemingly content with Papa’s answer. Briefly, serum and cell supernatants were centrifuged at 3000×g for 15 min to remove cells and cell debris. Nemmeno le celebrità. 2006;3:3.22. The purified 5T33MM-released exosomes were characterized by scanning electron microscopy (SEM), Zetasizer and Western blotting analysis. To validate the reliability of the p5 peptide in targeting the 5T33MM-released exosomes, we used an immuno-capture approach based on anti-CD63 decorated streptavidin magnetic nanoparticles (SMNPs) to trap exosomes [27]. 5T33MM–derived exosomes were equally detected using anti-mouse IgG or FITC-conjugated p5 peptide, while undetected when stained with control peptide, or left unstained (Fig. Cell Host Microbe. PubMed  performed the workflow illustration; F.M. B-cells from MM patient and healthy donor were isolated by negative selection from whole blood using RosetteSep Human B Cell Enrichment Cocktail [Stem Cell Technology, Vancouver, Canada], as previously described [26]. In particular, we identified peptide binders of the Ig-BCR idiotypic determinants (hereafter named “Id-peptides”) that are expressed on the surface of the A20 murine B-cell lymphoma, which revealed to be sensitive tools for in vivo tumor detection and tumor-specific delivery of radionuclides, fluorophores, siRNAs and nanoparticles [23]. Among 5T33MM serum-derived exosomes, we identified a p5 peptide-positive exosome sub-population 7 days after tumor inoculation, which increased in a time-dependent manner until death (Fig. PubMed  DNA fragment codifying the peptide ligand was amplified by PCR and sequenced to get the primary structure of amino acid sequence for the peptide synthesis. 2017;8(3):5179–95. The p5 peptide recognized specifically the 5T33MM-released exosomes. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. ELISA was performed to select ligands with distinct affinities for their cognate Ig-BCR. Ultimately, 3 cycles of panning were performed. 2013;14(8):793–803. 2013;8(9):e75054. Methods Mol Biol. 2015;10(3):e0117495. Exosomes are vesicles of 30–130 nm in diameter released by different cell types and detectable in all biological fluids [7] and supernatants of cultured cells [8]. The random peptide insert was flanked by a pair of cysteines resulting in cyclized peptide exposed on M13 coat protein. 5T33MM Igs-interacting phages were eluted with 0.2 M glycine-HCl (pH 2.2, 1 mg/mL BSA) followed by the addition of neutralizing solution (1 M Tris-HCl pH 9.1). CAS  Colombo M, Raposo G, Théry C. Annu Rev Cell Dev Biol. Danilo Sacco, 47 anni, 19 anni come cantante dei Nomadi, un primo posto in classifica e un infarto alle spalle, in queste settimane sta «rivedendo» la sua vita. 3a). Taddlr ha fatto una lista dei 35 attori più, Un sacco di celebrità non sa più cosa fare con i propri soldi. The Ph.D.-C7C Phage Display Peptide Library kit was purchased from New England Biolabs [NEB, Ipswich, Massachusetts, US]. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. A third group of 10 un-injected mice were used as negative control. Danilo Sacco (Agliano Terme, 6 giugno 1965) è un musicista e cantante italiano. 2b). For peptide co-localization with the BCR complex, 5T33MM cells (106 cells/ml) were stained with FITC-conjugated peptides [10 μg/ml) and goat anti-mouse IgG-Alexa fluor 568 [Thermo Fisher]. Leggi: 20 celebrità che non credevi fossero fumatori, Top 55 celebrità fumatrici più scandalose, Top 50 Foto più Scioccanti Fatte a Celebrità Senza Trucco, Top 35 degli Attori più Ricchi del Mondo – Patrimonio Attuale, Top 35 tatuaggi più folli delle celebrità, Top 35 delle Attrici più Ricche del Mondo. b Size distribution of the exosomes population derived from 5T33MM cells using dynamic light scattering (Zetasizer Nano S, Malvern Instruments). Exosomes were then purified from the supernatant of cultured 5T33MM, A20 and IM9 cells and from serum of MM patient and healthy donor. Flow cytometry of purified exosomes derived from cultured B-cells. Percentage of p5-FITC positive TDEs population after tumor cells injection. According to the Kaplan–Meier survival curve the fatal outcome of mice progressively occurred between 21 and 40 days post cells injection (Fig. In vitro characterization of exosome preparations. 2013;27:2. Saunderson SC, Schuberth PC, Dunn AC, Miller L, Hock BD, MacKay PA, Koch N, Jack RW, McLellan AD. The p5 peptide bound to the 5T33MM cell surface and also localized inside the cells (Fig. 5T33MM-engrafted mice develop a highly aggressive MM form, presenting biological and genetic characteristics similar to the human disease, and thus it represents one of the most reliable MM preclinical model [25]. 2008;180(12):8146–52. Article  The percentage of p5-FITC positive TDEs population in all mice is reported in the Additional file 1: Table S1. Gutzeit C, Nagy N, Gentile M, Lyberg K, Gumz J, Vallhov H, Puga I, Klein E, Gabrielsson S, Cerutti A, Scheynius A. J Immunol. The specific binding of peptides to the 5T33MM sIgG was analyzed by ELISA, as follows. The exosomes sample (10 μl) was spread, evaporated by using a vacuum concentrator at 30 °C, and analyzed by scanning electron microscopy [ESEM Quanta 400 instrument; FEI]. and G.S. Protein bands were detected using X-ray film and enhanced chemiluminescence reagent [GE Healthcare]. 2012;13(5):5674–99. PubMed  1. De Beule N, De Veirman K, Maes K, De Bruyne E, Menu E, Breckpot K, De Raeve H, Van Rampelbergh R, Van Ginderachter JA, Schots R, Van Valckenborgh E, Vanderkerken K. J Pathol. Although several methods have been developed for exosome purification, none of them clearly distinguish between normal and tumor-derived exosomes (TDEs), or avoid contamination by shed membrane vescicles [16]. Muller L, Mitsuhashi M, Simms P, Gooding WE, Whiteside TL. In this context, there is an urgent need to develop novel diagnostic approaches allowing the non-invasive early detection of tumor growth and the efficient monitoring of tumor progression [3]. Furthermore, compared with the standard exosome purification and immuno-capturing systems, the advantage of our method relies on the use of fluorescent-labeled Id-peptides coupled to SMNPs, which represent a more powerful and sensitive tool for tumor detection. Nat Cell Biol. Exosomes were purified from serum samples, conjugated with SMNPs and analyzed by flow cytometry using the FITC-conjugated p5 peptide. The specific Id-peptide binding to 5T33MM was verified by testing other B-cells, including the IM9 and A20 cell lines and primary B cells from a multiple myeloma patient and healthy donor (Additional file 1: Figure S1). helped in the conduction of the research plan and assisted with the data analysis; V.D. designed and conducted the research, analyzed the data, and wrote the manuscript; S.M. After brief vortex, samples were stored 30 min at 4 °C and then centrifuged at 1500 x g for 30 min at room temperature. Yao J, Yang M, Duan Y. Chem Rev. c Representative confocal images of 5T33MM cells labeled with FITC-conjugated p5 peptide (green), stained with the APC-conjugated anti-mouse IgG antibody (white), and DAPI (blue). Muratori C, Cavallin LE, Krätzel K, Tinari A, De Milito A, Fais S, Mesri EA, Superti F, Baur AS. In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. © 2020 BioMed Central Ltd unless otherwise stated. By scanning electron microscopy (SEM), the exosomes had a rounded shape (30–120 nm diameter) with some agglomerations occurring in the drying process during sample preparation (Fig. The screening of phage displayed library was performed using the bait 5T33MM Igs, as previously reported [22]. Part of To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. Workflow of the experimental design. è giocatore di football nel 2020 ha avuto successo per Real Madrid. c B-cells from the blood of multiple myeloma patient and 5T33MM cells as well as purified exosomes from patient serum or 5T33MM cell supernatant were lysed in RIPA buffer, separated by SDS-PAGE, and analyzed by Western blotting using the indicated antibodies. These results indicated that analysis of serum MM-released exosomes allowed an earlier detection of MM growth compared to the conventional measure of paraprotein. ... L’unico modo fare un bebè è devi essere solo con la tua moglie e essere molto intimo è amare l’un l’altro. Taddlr ha composto una lista dei 55 fumatori famosi più scandalosi. Article  Indeed, recent evidence demonstrated the utility of microvesicles in detecting relapse weeks before existing clinical tests, highlighting the sensitivity and capacity for microvesicles in monitoring disease progression and minimal residual disease in myeloma patients [6]. 2014;192(12):5852–62. assisted with the confocal microscopy analysis; P.C. Since this analysis was conducted on focal planes of non-permeabilized cells, the intracellular localization of the p5 peptide was a likely consequence of the BCR-mediated internalization. works at the SEM experiments; D.M. MM remains largely incurable due to the rapid development of aggressive, drug-resistant phenotypes [2]. All subsequent washing and incubations were carried out in the same buffer on ice. 2015;125(20):3076–84. After washing, membranes were hybridized with an alkaline phosphatase-conjugated anti-mouse IgG [Sigma Aldrich, Saint Louis, Missouri, US] (dilution 1:5000) and then washed 6 times. By flow cytometry, we analyzed the specific binding of FITC-conjugated p5 peptide to the 5T33MM, A20 and IM9 cells expressing the surface Ig-BCR. Iaccino, E., Mimmi, S., Dattilo, V. et al. E, Chi è l’attore più ricco di Hollywood? Google Scholar. To visualize the binding of the p5 peptide to the surface Igs, we performed confocal microscopy of 5T33MM cells stained with the FITC-conjugated p5 peptide and the anti-mouse IgG detecting the surface Igs. An irrelevant peptide (CGGNGPGLC) was included as a negative control (pCNT). Further, this analysis provided the evidence of similarity between murine and human multiple myeloma derived-exosomes in terms of IgG expression (Fig. 3c). PubMed Central  5b). Cite this article. The size of the exosomes-functionalized SMNPs (9.1 μm of diameter) supported the use of flow cytometric detection. Briefly, the streptavidin-conjugated beads [Thermo Fisher, Waltham, Massachusetts, US] were coated with 5T33MM Igs and incubated with 1 × 1011 phages overnight at 4 °C. 2c). b Concentration-dependent binding of FITC-conjugated p5 peptide and relative control peptide to 5T33MM cells, as measured by flow cytometry. Molecular Cancer PLoS One. Terms and Conditions, Aprirà i due concerti di Villastrada il duo Matteo Moretti e Simone Andreoli, a seguire Danilo Sacco in Trio. Danilo: Moglie, Amore, Amici e Famiglia Chi ha incontrato Danilo nel 2020? Per alcune sapete già che, Nessuno è perfetto. Springer Nature. Sul palco si esibiranno DANILO SACCO voce, Andrea Mei tastiere e Valerio Giambelli chitarra. J Cell Biol. Front/side scattering (FSC/SSC) plot of RedExo-labeled and un-labeled exosomes is shown in Fig. Salem KZ, Moschetta M, Sacco A, Imberti L, Rossi G, Ghobrial IM, Manier S, Roccaro AM. La consegna delle Targhe Tenco e dei Premi Tenco andrà in onda in TV, Taylor Swift, venduti per 300 milioni di dollari i diritti dei primi sei album, Random conquista il platino con “Sono un brano ragazzo un po’ fuori di testa”, Gioia social per i Ferragnez dopo la nomina per l’Ambrogino D’Oro “Squadra bellissima”, Iva Zanicchi torna a casa “Grazie ai medici che mi hanno assistita”, Musictory allows YouTube to serve content, including advertisements. Tumor progression and serum exosomes production in tumor-engrafted mice. Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. helped in the data analysis; A.D.L., E.V., T.G., and S.C. assisted with the mouse experiments; A.P., F.A., A.L., D.M, and G.F. worked at the biochemical analysis; I.Q. Sci Rep. 2016;6:20254. Mangini M, Iaccino E, Mosca MG, Mimmi S, D'Angelo R, Quinto I, Scala G, Mariggiò S. Oncotarget. At day 0 and each 7 days, blood samples were collected by retro-orbital bleeding for measuring the serum MM-released exosomes and paraprotein levels. Si riempie il bicchiere anche la voce dei Nomadi. Monitoring MM progression is a crucial step for determining the stage of disease and choosing the most appropriate therapy. Isolated exosomes from serum or cell supernatants were suspended in 1 ml of 1X PBS. By ELISA, the serum paraprotein was detected 21 days after tumor inoculation (Fig. First identified in the mid-80s [4] and initially classified as unfunctional “garbage bags” containing unwanted cellular constituents, exosomes represent a promising tool for novel diagnostic options in the diagnosis of malignant diseases [5]. Article  Mimmi S, Vecchio E, Iaccino E, Rossi M, Lupia A, Albano F, Chiurazzi F, Fiume G, Pisano A, Ceglia S, Pontoriero M, Golino G, Tassone P, Quinto I, Scala G, Palmieri C. Leukemia. After removing the supernatant, the pellet was suspended in nuclease-free water. Marimpietri D, Petretto A, Raffaghello L, Pezzolo A, Gagliani C, Tacchetti C, Mauri P, Melioli G. Pistoia V. PLoS One. The authors declare that they have no competing interests. Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia,”, Catanzaro, Italy, Enrico Iaccino, Selena Mimmi, Vincenzo Dattilo, Fabiola Marino, Patrizio Candeloro, Antonio Di Loria, Antonio Pisano, Francesco Albano, Eleonora Vecchio, Simona Ceglia, Gaetanina Golino, Antonio Lupia, Giuseppe Fiume, Ileana Quinto & Giuseppe Scala, Stem Cell and Cellular Therapy Laboratory, G. Gaslini Institute, Genoa, Italy, You can also search for this author in Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. supervised the research plan and data analysis, and reviewed the manuscript. Google has many special features to help you find exactly what you're looking for. Google Scholar. Lasser C, Alikhani VS, Ekstrom K, Eldh M, Paredes PT, Bossios A, Sjostrand M, Gabrielsson S. Lotvall J and Valadi H. J Transl Med. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Exosome aliquots (500 μl) were labeled with 50 μl of 10X Exo-Red [SBI] according to manufacturer’s instructions. 5c). For Western blot analysis, the exosomes were lysed in reducing sample buffer [0.25 M Tris–HCl (pH 6.8), 40% glycerol, 8% SDS, 5% 2-mercaptoethanol and 0.04% bromophenol blue] and boiled for 10 min at 95 °C. Google Scholar. 4). Google Scholar. 2011;9:9. As controls, the FITC-conjugated p5 peptide did not detect the exosomes released from the human IM9 multiple myeloma and murine A20 B-lymphoma cells (Fig. Privacy Questo giocatore di football enigmatico proveniente da Bicas, Brazil ha un corpo atletico & tipo di viso ovale. 1985;101:942–8. Correspondence to Search the world's information, including webpages, images, videos and more. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression. 2017;241(4):534–46. Front/side scattering of exosome-bound SMNPs (External plot); Exo-Red-stained exosomes incubated with FITC-conjugated anti-IgG (left plots); Exo-Red-stained exosomes incubated with FITC-conjugated p5 peptide (central plots); Exo-Red-stained exosomes incubated with FITC-conjugated pCNT (right plots). In conclusion, we describe the development of a method allowing the rapid and simple detection of MM-released exosomes using the Id-peptide binders of the Igs expressed by tumor B-cells. Figlio di padre (?) Figure S1. Associazione Italiana per la Ricerca sul Cancro to G.S. Chiudendo questo banner, scrollando la pagina acconsenti all'uso dei cookie. Tuccillo FM, Palmieri C, Fiume G, de Laurentiis A, Schiavone M, Falcone C, Iaccino E, Galandrini R, Capuano C, Arra C, Barbieri A, Dal Piaz F, Venzon D, Bonelli P, Buonaguro FM, Scala I, Mallardo M, Quinto I, Scala G. Mol Cancer Ther. Enrico Iaccino or Ileana Quinto. 2014;114(12):6130–78. d Flow cytometric analysis of Red-Exo-stained exosomes derived from serum of a representative A20-engrafted mouse incubated with FITC-conjugated p5 peptide. L ‘evento è stato organizzato da Massimo Nizzoli attuale presidente del teatro di … In the last few years, we successfully validated the screening of random peptide libraries (RPLs) as a method to identify peptides binders of soluble immunoglobulins (Igs) [19] transmembrane receptors [20, 21] and biomaterials [22]. (PRIN project 2012CK5RPF; PRIN project 2012CK5RPF_002); Grant POR CALABRIA FSE 2007/2013 PON01/00862 to GS; Ministero della Salute Ministero della Salute to G.S. Based on specificity, flexibility, cost effectiveness and modularity, our capture system is ideal for novel non-invasive applications in personalized medicine and could be easily extended to other B-cell malignancies. By using this website, you agree to our Proteins were resolved by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), transferred to poly-vinylidene fluoride membranes, blocked with 5% non-fat powdered milk in PBS-T (0.5% Tween-20) and probed with anti-mouse CD63, anti- mouse CD81, or anti-mouse IgG antibodies. J Immunol. The p5 peptide showed a concentration-dependent specific reactivity to the cognate antibody, and did not react against an A20 secreted IgG or polyclonal mouse Igs (Fig. Confirmation of the size homogeneity of vesicles was verified using a Zetasizer Nano ZS90 (Fig. Wells were extensively washed and coated with the anti-mouse IgG (Fc-specific) alkaline phosphatase-conjugated [Sigma Aldrich] for 1 h at 37 °C, incubated with the alkaline phosphatase substrate [Sigma Aldrich], and analyzed by an ELISA reader at 405 nm [Labsystems multiscan MS]. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). (Grant IG-2009- 9411; IG Grant 2012–13,388); Ministero dell’Istruzione, dell’Università e della Ricerca to G. S and I.Q. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Tumor-derived exosomes and paraprotein levels in peripheral blood were monitored every 7 days up to 35 days post-inoculation, In vitro binding of selected Id-peptides to the 5T33MM-Ig. Igs were purified from the culture supernatants by using the Mab Trap™ antibody purification Kit [GE Healthcare, Little Chalfont, UK], according to the manufacturer’s instructions. To this end, the biotinylated-p5 peptide was incubated with 5T33MM-Igs coated plates at different concentrations, and revealed with streptavidin-conjugated alkaline phosphatase. E.I. 5T33MM, A20 and IM9 B cell lines bear surface Igs that are secreted in the culture medium. Exosomes contain a wide range of RNA and proteins, playing an important role in cell-to-cell communication [9].
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