“Oh,” Danilo said, looking down at his cards, seemingly content with Papa’s answer. 2016;18(1):25–32. 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. è giocatore di football nel 2020 ha avuto successo per Real Madrid. MM remains largely incurable due to the rapid development of aggressive, drug-resistant phenotypes [2]. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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]. 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). 19 ago 2009 - Metti una sera prima di cena, ad Agliano Terme, nido di Danilo Sacco. 2007;9:654–9. 3c). A total number of 10 mice per group were respectively injected with 106 cells of 5T33MM multiple myeloma cells or A20 B-lymphoma cells. In particular, exosomes are involved in the regulation of the immune response, antigen presentation [10], tumor survival [11], cell migration [12], tumor invasion [13], cell differentiation and angiogenesis [14]. 2014;192(12):5852–62. Lasser C, Alikhani VS, Ekstrom K, Eldh M, Paredes PT, Bossios A, Sjostrand M, Gabrielsson S. Lotvall J and Valadi H. J Transl Med. S. M. was supported by a fellowship from Associazione Italiana per la Ricerca sul Cancro (AIRC, FIRC). Salem KZ, Moschetta M, Sacco A, Imberti L, Rossi G, Ghobrial IM, Manier S, Roccaro AM. After removing the supernatant, the pellet was suspended in nuclease-free water. 2017;241(4):534–46. Nemmeno le celebrità. 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. Li J, Liu K, Liu Y, Xu Y, Zhang F, Yang H, Liu J, Pan T, Chen J, Wu M, Zhou X, Yuan Z. Nat Immunol. 4a. CAS  5a). The Ph.D.-C7C Phage Display Peptide Library kit was purchased from New England Biolabs [NEB, Ipswich, Massachusetts, US]. https://doi.org/10.1186/s12943-017-0730-8, DOI: https://doi.org/10.1186/s12943-017-0730-8. Causa F, Della Moglie R, Iaccino E, Mimmi S, Marasco D, Scognamiglio PL, Battista E, Palmieri C, Cosenza C, Sanguigno L, Quinto I, Scala G, Netti PA. J Colloid Interface Sci. Exosomes contain a wide range of RNA and proteins, playing an important role in cell-to-cell communication [9]. Théry C, Amigorena S, Raposo G. Clayton A. Curr Protoc Cell Biol. Yao J, Yang M, Duan Y. Chem Rev. a Scanning electron microscopy (SEM). Flow cytometry analysis of purified exosomes derived from different cell sources (DOC 570 kb). Part of Exosomes were purified from serum samples, conjugated with SMNPs and analyzed by flow cytometry using the FITC-conjugated p5 peptide. 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. Google Scholar. 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]. 3a). California Privacy Statement, 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). 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. 2006;3:3.22. c Flow cytometric analysis of Red-Exo-stained exosomes derived from serum of a representative 5T33MM-engrafted mouse incubated with FITC-conjugated p5 peptide. The supernatant was transferred to a sterile vessel and the recommended volume of ExoQuick solution or ExoQuick-TC™ were added to the bio-fluids. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosomes were isolated from 250 μL of blood and 10 mL of cell supernatant respectively using ExoQuick solutions [System Biosciences – SBI, Palo Alto, California, US] and ExoQuick-TC™ [System Biosciences], according to the manufacturer’s protocol. Article  2008;180(12):8146–52. 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. Springer Nature. Questo sito web utilizza cookies di profilazione di terze parti per migliorare la tua navigazione. 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. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Serum-derived exosomes from A20 harboring mice were also purified and used as control (Fig. 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. 2012;13(5):5674–99. 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]. 4). 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]. The labeled exosome pellet was suspended in 500 μl 1X PBS and stained with CD63-coupled magnetic beads provided by SBI’s Exo-Flow IP kit [SBI] and with the FITC-conjugated peptides. (Grant IG-2009- 9411; IG Grant 2012–13,388); Ministero dell’Istruzione, dell’Università e della Ricerca to G. S and I.Q. The percentage of p5-FITC positive TDEs population in all mice is reported in the Additional file 1: Table S1. In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. E.I. PubMed Central  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. Cell Host Microbe. 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]. 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]. PubMed  Article  PubMed  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. Nat Cell Biol. Even if the mechanism of expression remains not completely defined, it is worthwhile that MM-released exosomes constitutively express on their surface the immunoglobulin of B-cell receptor (Ig-BCR) derived from the parental tumor B-cell, and thus they can be reliable tumor markers [17, 18]. The p5 peptide recognized 5T33MM target cells in a dose-dependent manner, while the pCNT peptide did not (Fig. The random peptide insert was flanked by a pair of cysteines resulting in cyclized peptide exposed on M13 coat protein. To this end, the biotinylated-p5 peptide was incubated with 5T33MM-Igs coated plates at different concentrations, and revealed with streptavidin-conjugated alkaline phosphatase. 1. 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. 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. Certo, in television, nelle riviste e nei film – con, Potreste chiedervi dove vivono le celebrità con così tanto denaro nel loro conto in banca. 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]. 2015;125(20):3076–84. PubMed Central  After extensive washing, cells were mounted under cover slip, and visualized by confocal microscopy [Leica TC-SP2]. 2016;30(12):2419–22. Isolated exosomes from serum or cell supernatants were suspended in 1 ml of 1X PBS. Briefly, serum and cell supernatants were centrifuged at 3000×g for 15 min to remove cells and cell debris. Marimpietri D, Petretto A, Raffaghello L, Pezzolo A, Gagliani C, Tacchetti C, Mauri P, Melioli G. Pistoia V. PLoS One. SMNPs were decorated with biotinylated anti-CD63, incubated with RED-EXO-labeled exosomes, and analyzed for the binding of FITC-conjugated Id-peptides by flow cytometry. Multiple myeloma (MM) is a clonal B-cell malignancy accounting for more than 10% of hematologic cancers, and is characterized by the aberrant expansion of bone marrow plasma cells releasing a high level of monoclonal immunoglobulin (mIg) in the blood, so called paraprotein [1]. Danilo Sacco (Agliano Terme, 6 giugno 1965) è un musicista e cantante italiano. Being the IgBCR expressed on the surface of tumor B cells as well as tumor-derived exosomes, our methodology could be extended to the most common B cell neoplasia with potential application for monitoring the minimal residual disease. Manage cookies/Do not sell my data we use in the preference centre. 2011;9:9. The levels of serum IgG2b from the blood (250 μL) of tumor-engrafted mice or control mice were measured using the Easy-Titer™ Mouse IgG Assay Kit [Thermo Fisher] according to manufacturer’s instructions. performed the workflow illustration; F.M. Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To this end, we measured the tumor growth and serum MM-released exosomes in vivo in the 5T33MM murine model [24]. b Size distribution of the exosomes population derived from 5T33MM cells using dynamic light scattering (Zetasizer Nano S, Malvern Instruments). 2013;8(9):e75054. 5b). Exosomes were then purified from the supernatant of cultured 5T33MM, A20 and IM9 cells and from serum of MM patient and healthy donor. Exosome aliquots (500 μl) were labeled with 50 μl of 10X Exo-Red [SBI] according to manufacturer’s instructions. After three rounds of affinity selection, single phage clones were purified and tested by enzyme-linked immunosorbent assay (ELISA) for binding to the 5T33MM Igs or control Igs, as previously described [26]. Purified exosomes were also analyzed by Western blotting using antibodies against typical exosomal markers, such as CD81, CD63, including anti-IgG antibody to verify the IgG expression on TDEs (Fig. La moglie Chiara serve un frizzantino fresco. Reflecting the genomic and proteomic profile of their parental cells, circulating serum exosomes are potential biomarkers in predicting cancer burden with relevant impact for personalized therapy [15]. Interessante. Iaccino, E., Mimmi, S., Dattilo, V. et al. Muratori C, Cavallin LE, Krätzel K, Tinari A, De Milito A, Fais S, Mesri EA, Superti F, Baur AS. Google Scholar. Lemaire M, Deleu S, De Bruyne E, Van Valckenborgh E, Menu E, Vanderkerken K. Adv Cancer Res. b Determination of serum paraprotein (IgG2b) concentration in 5T33MM- or A20-engrafted mice and control mice measured by ELISA. volume 16, Article number: 159 (2017) The production of IgG-bearing exosomes was similar in the three cell lines. Article  CAS  In vitro characterization of exosome preparations. The purified 5T33MM-released exosomes were characterized by scanning electron microscopy (SEM), Zetasizer and Western blotting analysis. PubMed  According to the Kaplan–Meier survival curve the fatal outcome of mice progressively occurred between 21 and 40 days post cells injection (Fig. Beads were extensively washed with PBS supplemented with 0.05% Tween-20 to remove unbound phage. 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. The authors declare that they have no competing interests. Synthesized peptides were purchased from Caslo Laboratory ApS [Caslo, Kongens Lyngby, DE]. 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]. Based on the highest affinity binding to 5T33MM Igs, the insert amino acid sequence of phage clone 5 (CIGNSNTLC) was used for large-scale synthesis of the p5 peptide in order to evaluate the binding properties outside of the phage context. 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). 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. 2011;110:19–42. Methods Mol Biol. All authors read and approved the final manuscript. 4). 2016;49(1):33–50. Schiavone M, Fiume G, Caivano A, de Laurentiis A, Falcone C, Masci FF, Iaccino E, Mimmi S, Palmieri C, Pisano A, Pontoriero M, Trovato M, Rafay J, Ferko B, Morsica G, Poli G, Quinto I, Pavone V, de Berardinis P, Scala G. Int J Mol Sci. 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. CAS  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]. 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. ELISA was performed to select ligands with distinct affinities for their cognate Ig-BCR. Google Scholar. Protein bands were detected using X-ray film and enhanced chemiluminescence reagent [GE Healthcare]. Saunderson SC, Schuberth PC, Dunn AC, Miller L, Hock BD, MacKay PA, Koch N, Jack RW, McLellan AD. 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). and G.S. 4b-e). 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. 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]. Il 15-7-1991 Danilo (soprannome: Danilo) è nato a Bicas, Brazil. helped in the conduction of the research plan and assisted with the data analysis; V.D. 2014;30:255–89. 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. d Flow cytometric analysis of Red-Exo-stained exosomes derived from serum of a representative A20-engrafted mouse incubated with FITC-conjugated p5 peptide. 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 Colombo M, Raposo G, Théry C. Annu Rev Cell Dev Biol. Chiudendo questo banner, scrollando la pagina acconsenti all'uso dei cookie. works at the SEM experiments; D.M. 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). 2017;8(3):5179–95. 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. 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. Figlio di padre (?) Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Enrico Iaccino or Ileana Quinto. Privacy assisted with the confocal microscopy analysis; P.C. Sul palco si esibiranno DANILO SACCO voce, Andrea Mei tastiere e Valerio Giambelli chitarra. PubMed  PubMed  The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cookies policy. If you continue to use this site we will assume that you are ok with it. Muller L, Mitsuhashi M, Simms P, Gooding WE, Whiteside TL. Workflow of the experimental design. Immunoreactive phage clones were detected by BCIP/NBT premixed substrate [Thermo Fisher]. The analysis was performed using a Leica TCS SP2 confocal microscope at 40X magnification. http://creativecommons.org/licenses/by/4.0/, http://creativecommons.org/publicdomain/zero/1.0/, https://doi.org/10.1186/s12943-017-0730-8. For in vivo analysis, 5T33MM cells (1 × 106) were intravenously injected in C57BL/KaLwRij mice (10 females at 8 weeks of age). Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. 2010;116(2):226–38. Peptide binding to A20 secreted Igs and polyclonal mouse Igs were included as controls. 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. Sci Rep. 2016;6:20254. The p5 peptide recognized specifically the 5T33MM-released exosomes. 2013;389(1):220–9. Confirmation of the size homogeneity of vesicles was verified using a Zetasizer Nano ZS90 (Fig. Article  a Kaplan-Meier survival curves in 5T33MM-engrafted mice (n = 10), A20-engrafted mice (n = 10) and un-grafted control mice (n = 10). Figure S1. supervised the research plan and data analysis, and reviewed the manuscript. Taddlr ha composto una lista dei 55 fumatori famosi più scandalosi. Molecular Cancer statement and 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. An irrelevant peptide (CGGNGPGLC) was included as a negative control (pCNT). The experimental plan undertaken is shown in Fig. 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. 2b). The authors thank Prof. Karin Vanderkerken (Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, Brussels, Belgium) for the 5T33MM tumor cells, and Dr. Donato Cosco (Department of Health Sciences, University of Catanzaro “Magna Græcia” Catanzaro, Italy) for the assistance with the Zetasizer. The screening of phage displayed library was performed using the bait 5T33MM Igs, as previously reported [22]. 2015;10(3):e0117495. Danilo Sacco Articolo 2020 ⁓ Di Più Check-out Danilo Sacco riferimento- potresti anche essere interessato a Danilo Sacco Nomadi e così via Danilo Sacco Picchia Moglie . Krishnan SR, Luk F, Brown RD, Suen H, Kwan Y, Bebawy M. Neoplasia. Exosomes were purified from supernatants of the murine 5T33MM and human IM9 multiple myeloma cells, and the murine A20 B-lymphoma cells. e madre(?) The 5T33MM cell culture supernatant was filtered (0.22 μm) and the exosomes were purified using the ExoQuick-TC™. 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. 2a). Questo giocatore di football enigmatico proveniente da Bicas, Brazil ha un corpo atletico & tipo di viso ovale. 2c). Cite this article. 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]. 2014;13(3):752–62. 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. Synthetic peptides corresponding to the peptide insert of phage clones were assayed for their antigenic properties out of the phage context. Aprirà i due concerti di Villastrada il duo Matteo Moretti e Simone Andreoli, a seguire Danilo Sacco in Trio. 1985;101:942–8. 2016;1464:25–34. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. Cells were incubated with 20 μg/ml of FITC-conjugated peptides for 10 min on ice, then washed twice, and analyzed by flow cytometry [FACSCalibur, BD Biosciences]. Google has many special features to help you find exactly what you're looking for. Moreau P, Attal M, Facon T. Blood. 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. 5d). Streptavidin coated 96 well plates were washed extensively and supplemented with biotin-conjugated peptides by 1 h-incubation at 37 °C; then, after washing and blocking with blocking solution (1X PBS, 0.05% Tween-20, 5% milk), aliquots of 5T33MM sIgG (1 μg/ml in blocking buffer) were added overnight at 4 °C.