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Ruta, Claudia. « La coltura in vitro per la conservazione della biodiversità orticola ». Italus Hortus 25 (2018) : 1–11. http://dx.doi.org/10.26353/j.itahort/2018.1.3690.
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Botto, A., S. Ungari, S. Riba, S. Ribero, C. Dadone, M. Mediago et R. Zolfanelli. « Coltura Cellulare E Test Di Chemiosensibilità in Vitro Nelle Neoplasie Vescicali Superficiali : Risultati Preliminari Dell'Applicazione Clinica ». Urologia Journal 58, no 5 (octobre 1991) : 570–72. http://dx.doi.org/10.1177/039156039105800520.
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Di Lullo, A. M., M. Scorza, F. Amato, M. Comegna, V. Raia, L. Maiuri, G. Ilardi, E. Cantone, G. Castaldo et M. Iengo. « An ex vivo model contributing to the diagnosis and evaluation of new drugs in cystic fibrosis ». Acta Otorhinolaryngologica Italica 37, no 3 (juin 2017) : 207–13. http://dx.doi.org/10.14639/0392-100x-1328.
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La fibrosi cistica (FC) è una malattia autosomica recessiva causata da mutazioni nel gene CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Finora sono state descritte circa 2000 mutazioni, ma per la maggior parte di esse è difficile definirne leffetto senza complesse procedure in vitro. Abbiamo effettuato il campionamento (mediante brushing), la cultura e lanalisi di cellule epiteliali nasali umane (HNEC) utilizzando una serie di tecniche che possono aiutare a testare leffetto delle mutazioni CFTR. Abbiamo eseguito 50 brushing da pazienti FC e controlli, e in 45 casi si è ottenuta una coltura positiva. Utilizzando cellule in coltura: i) abbiamo dimostrato lespressione ampiamente eterogenea del CFTR nei pazienti e nei controlli; ii) abbiamo definito leffetto di splicing di una mutazione sul gene CFTR; iii) abbiamo valutato lattività di gating di CFTR in pazienti portatori di differenti mutazioni; iv) abbiamo dimostrato che il butirrato migliora in modo significativo lespressione di CFTR. I dati provenienti dal nostro studio sperimentale dimostrano che luso del modello ex-vivo di cellule epiteliali nasali è un importante e valido strumento di ricerca e di diagnosi nella studio della FC e può anche essere mirato alla sperimentazione ed alla verifica di nuovi farmaci. In definitiva, in base ai nostri dati è possibile esprimere le seguenti conclusioni: 1) il prelievo delle cellule epiteliali nasali mediante brushing è applicabile senza alcuna anestesia ed è ben tollerato da tutti i pazienti affetti da FC (bambini e adulti), è scarsamente invasivo e facilmente ripetibile, è anche in grado di ottenere una sufficiente quantità di HNECs rappresentative, ben conservate, idonee allo studio della funzionalità di CFTR; 2) la conservazione delle cellule prelevate è possibile fino a 48 ore prima che si provveda allallestimento della coltura e ciò permette di avviare studi multicentrici con prelievi in ogni sede e quindi di ottenere una ampia numerosità campionaria; 3) la coltura di cellule epiteliali nasali può essere considerata un modello adatto a studiare leffetto molecolare di nuove mutazioni del gene CFTR e/o mutazioni specifiche di pazienti carriers dal significato incerto; 4) il modello ex-vivo delle HNECs consente inoltre di valutare, prima dellimpiego nelluomo, leffetto di farmaci (potenziatori e/o correttori) sulle cellule di pazienti portatori di mutazioni specifiche di CFTR; tali farmaci possono modulare lespressione genica del canale CFTR aprendo così nuove frontiere terapeutiche e migliori prospettive di vita per pazienti affetti da una patologia cronica come la Fibrosi Cistica; 5) la metodologia da noi istituita risulta essere idonea alla misura quantitativa, mediante fluorescenza, dellattività di gating del canale CFTR presente nelle membrane delle cellule epiteliali nasali prelevate da pazienti portatori di differenti genotipi; in tal modo è possibile individuare: a) pazienti FC portatori di 2 mutazioni gravi con unattività < 10% (in rapporto ai controlli -100%), b) soggetti FC portatori contemporaneamente di una mutazione grave e di una lieve con unattività tra 10-30%, c) i cosiddetti portatori carriers- eterozigoti - con unattività tra 40-70%. In conclusione la possibilità di misurare lattività del canale CFTR in HNECs fornisce un importante contributo alla diagnosi di FC, mediante individuazione di un cut-off diagnostico, ed anche alla previsione della gravità fenotipica della malattia; quindi quanto rilevabile dalla misura del suddetto canale permette di prospettare per il futuro la possibilità di valutare meglio i pazienti per i quali il test del sudore ha dato risultati ambigui (borderline o negativi). La metodica da noi sperimentata consente anche di monitorare i pazienti durante il trattamento farmacologico, valutando in tal modo i reali effetti delle nuove terapie.
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Di Pietro, Maria Luisa, et Roberta Minacori. « Qual è il rischio delle tecniche di fecondazione artificiale ? » Medicina e Morale 47, no 3 (30 juin 1998) : 465–97. http://dx.doi.org/10.4081/mem.1998.832.
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L’articolo mette a fuoco i rischi che, o per l’imperizia dell’operatore, o per le procedure previste dalla tecnica di fecondazione, sono responsabili della morbilità e mortalità della donna e/o del nascituro. Uno dei rischi per la donna è legato alla stimolazione ovarica che si attua con una induzione farmacologica e che provoca la cosiddetta “iperstimolazione ovarica” e tumori della mammella e dell’ovaio.
Altri rischi sono legati alle complicanze delle procedure di fecondazione artificiale e che riguardano soprattutto la fase di recupero delle ovocellule, la coltura in vitro, il trasferimento dei gameti e degli embrioni nelle vie genitali della donna. Il prelievo degli ovociti viene eseguito sotto controllo ecografico con rischio di dolori pelvici o addominali, infezioni ed emorragie, mentre il trasferimento dei gameti si esegue con la laparoscopia con complicanze legate all’anestesia. Il primo rischio per l’embrione è che non arrivi a vita autonoma per mancato trasferimento nelle vie genitali della donna, il mancato attecchimento nell’utero, l’aborto spontaneo o provocato, la maggiore incidenza di morbilità o di mortalità perinatale.
Altri fattori negativi sono dovuti alla micromanipolazione dei gameti. Inoltre, dall’analisi della letteratura si evince che i nati da fecondazione artificiale presentano maggiori malformazioni congenite e una più elevata incidenza di prematurità. A tutti questi problemi si aggiunge l’inadeguata informazione fornita alle coppie che ricorrono alla fecondazione artificiale sui rischi, sugli effetti collaterali, sulle percentuali di successo.
5
Capuana, Maurizio. « Le colture in vitro per il fitorimedio ». Italus Hortus, no 24 (2018) : 15–28. http://dx.doi.org/10.26353/j.itahort/2017.3.1528.
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Botto, A., C. Bumma, S. Riba, S. Ungari et R. Zolfanelli. « Chemiosensibilità Del Carcinoma Renale Su Colture Cellulari in Vitro ». Urologia Journal 56, no 4 (août 1989) : 507–12. http://dx.doi.org/10.1177/039156038905600420.
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Vitali, G., L. Conte, S. Foddai et M. Nicoletti. « Colture in Vitro di Withania Somnifera Dunal (Solanaceae) Chemotipo Italiano ». Giornale botanico italiano 128, no 1 (janvier 1994) : 453. http://dx.doi.org/10.1080/11263509409437260.
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Canzi, M., P. Coral, T. Roggio, L. De Filippo et G. Panarello. « Valutazione clinico/morfologica di Amukine Med® e Braunol®, su CVC in spisilicone ». Giornale di Clinica Nefrologica e Dialisi 23, no 2 (24 janvier 2018) : 19–22. http://dx.doi.org/10.33393/gcnd.2011.1431.
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La gestione infermieristica riveste un ruolo importante per la sopravvivenza dell'accesso vascolare per emodialisi, soprattutto quando per la sua realizzazione sono stati utilizzati materiali protesici etcrologhi. Scopo di questo studio è di valutare in vitro e in vivo gli eventuali effetti collaterali e l'efficacia di due disinfettanti tra i più comunemente usati (ipoclorito di sodio allo 0,057 Amukine Med® e iodopovidone al 10% Braunol®,) per le medicazioni dei cateteri venosi centrali. Lo studio è stato effettuato da gennaio 2003 a gennaio 2004. In tale periodo abbiamo valutato in vitro mediante esame morfologico gli effetti sui cateteri incubati a breve e lungo termine nei 2 disinfettanti e in vivo l'incidenza di reazioni cutanee locali e la positività dell'esame colturale del tampone cutaneo, in 17 malati uremici con “Tesio cat®” Medcomp (spisilicone) come accesso vascolare per emodialisi. Non si sono notate differenze morfologiche significative nello studio in vitro tra i campioni trattati con i due disinfettanti. Il contatto prolungato dello spisilicone con Amukine Med e Braunol anche in ambiente libero non ha determinato alterazioni morfologiche della parete all'esame macro e microscopico. Nello studio in vivo, condotto su due gruppi composti da 10 pazienti nel gruppo Amukine Med e 7 pazienti in quello con Braunol, sono state effettuate 1088 medicazioni (640 con Amukine Med pari al 58,8% medicazioni totali e 448 con Braunol pari al 41,2% medicazioni totali) pari al 40,5% delle sedute dialitiche con ambo le tecniche di disinfezione. Dagli esami colturali (271 tamponi) in 71 casi è stata riportata crescita batterica; 68 Staphilococcus Epidermidis; 2 Escherichia Coli (gruppo Amuchina Med) 1 Pseudomonas Aeruginosa (gruppo Braunol). Negli ultimi 3 casi (1/68 mesi d'esposizione) era presente sepsi locale. Non si sono rilevate differenze nell'incidenza di infezioni locali o di effetti collaterali indotti dai due disinfettanti.
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Forni, C., A. Frattarelli et M. Grilli Caiola. « Studi Sulla Rigenerazione in Colture in Vitro di Fragaria X Ananassa Duch ». Giornale botanico italiano 130, no 1 (janvier 1996) : 387. http://dx.doi.org/10.1080/11263509609439622.
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De Angeli, S., A. Fandella, C. Gatto, S. Buoro, C. Favretti, G. L. Drago Ferrante et G. Anselmo. « Stromal cell and human prostatic epithelial cell in-vitro co-coltures : Growth and morphology ». Urologia Journal 63, no 1_suppl (janvier 1996) : 65–68. http://dx.doi.org/10.1177/039156039606301s16.
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A study was carried out on the effect of stroma-epithelium interaction on cellular growth and morphology in co-coltures of U285 prostatic epithelial cells with human prostatic and esophageal stromal cells and with murine fibroblasts of the 3T3-J2 line. The proliferation rate was determined by growth tests of neutral red and kenacid blue. Morphological observations were made under optical microscope on the same cultures used for the growth tests. Results highlighted a marked reduction in cellular growth in the co-cultures compared to control cultures, as well as the tendency of the stromal and epithelial cells to re-organise themselves in pseudo-acinous structures.
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Toscani, Denise, Carla Palumbo, Benedetta Dalla Palma, Marina Bolzoni, Marzia Ferretti, Paola Sena, Daniela Guasco, Eugenia Martella, Franco Aversa et Nicola Giuliani. « Myeloma-Induced Osteocyte Death Was Blunted By Proteasome Inhibitors Through The Modulation Of Autophagy ». Blood 122, no 21 (15 novembre 2013) : 3096. http://dx.doi.org/10.1182/blood.v122.21.3096.3096.
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Abstract Osteocytes are critical in the maintenance of bone integrity regulating bone remodeling through the cell death and autophagy, a cellular process stress-induced to prolong cell survival but when induced excessively can cause cell death. Recently we have demonstrated that an increased osteocyte death is involved in multiple myeloma (MM)-induced osteolysis. However the mechanisms involved in this process as well as the effect of the proteasome inhibitors able to stimulate bone formation are not known and have been investigated in this study. Firstly the effect of the proteasome inhibitors BOR and MG262 on osteocyte viability was evaluated in vitro in murine osteocytic cell line MLO-Y4 and in the human pre-osteocytic one HOB-01. Both cell lines were co-coltured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) RPMI8226 and JJN3, placed in a traswell insert. The treatment for 12-24 hours with (BOR) (2nM) and MG262 (10nM) significantly blunted MLO-Y4 and HOB-01 cell death. In addition, dexamethasone (DEX)-induced MLO-Y4 apoptosis, obtained at high doses (10-5-10-6 M), was reduced by the treatment with proteasome inhibitors. Interestingly, we found that PTH short-term treatment potentiated the in vitro effects of proteasome inhibitors on DEX-induced osteocyte death. To evaluate the presence of autophagy in osteocytes, we checked the expression of the autophagic marker LC3 both by confocal microscopy and western blot analysis in the co-colture system with MLO-Y4 and RPMI-8226. Prevalence of autophagic cell death and in a lesser extent apoptosis was observed in this system. BOR increased the basal level of LC3 indicating a pro-survival and protective function of autophagy against the BOR-induce stress. On the contrary, when cells undergo to a stronger stress such as in the presence of HMCLs or by treatment with high dose of DEX we found that both proteasome inhibitors BOR and MG262 blocked autophagic cell death in osteocytes. To translate our in vitro evidence in a clinical perspective, thereafter we performed a histological evaluation on bone biopsies of a cohort of 37 newly diagnosis MM patients 31 of them with symptomatic MM and 6 with smoldering MM (SMM). The 55% of patients with MM have evidence of osteolytic lesions at the X-rays survey. Bone biopsies were obtained at the diagnosis and after an average time of 12 months of treatment or observation. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections. A significant increase of the number of viable osteocytes was demonstrated in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase: +6% vs. +1.30%; p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability, as compared to those either treated without BOR (+11.6% vs. +1.3%, p=0.0019) or treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). A reduction of both osteocyte apoptosis and autophagy was demonstrated by TUNEL assays and confocal microscopy. On the other hand, any significant difference was not observed in patients treated with Thalidomide (THAL) or Immunomodulatory drugs (IMiDs) than in those untreated with these drugs (p= 0.7). A multiple regression non-parametric analysis showed that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas THAL/IMiDs as well as Zoledronic acid (ZOL) treatments have not (p=0.2). BOR also counterbalanced the negative effect of DEX treatment (p=0.035). Our data suggest that proteasome inhibitors blunted osteocyte cell death induced by MM cells and DEX through the modulation of the autophagy supporting their use to improve bone integrity in MM patients. Disclosures: Giuliani: Celgene Italy: Research Funding.
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Cappellini, Maria D., Ilaria V. Libani, Elisabetta Calzavara, Luisa Ronzoni, Raffaella Chiaramonte et Paola Comi. « Expression of Hes-1 (Notch-1 Effector) during “In Vitro” Normal Erythroid Differentiation. » Blood 106, no 11 (16 novembre 2005) : 4287. http://dx.doi.org/10.1182/blood.v106.11.4287.4287.
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Abstract Hematopoiesis involves highly regulated proliferation and differentiation during which a small number of multipotent stem cells give rise to differentiated progenies. In several developmental systems stem cells fate is influenced by soluble molecules acting via cell-cell interaction, including those mediate by the Notch receptor family. Members of Notch family transmembrane receptors are found on primitive hematopoietic precursors, suggesting a role for Notch signaling in mammalian blood cells development. Notch signaling regulates cell fate controlling asymmetric cell division during stem/progenitor cell differentiation. A previous study on K562 cell line showed that Notch signaling inhibits erythroid/megakaryocytic development by suppressing GATA-1 activity. Furthermore there are evidences that Notch is expressed in early murine erythroid precursors. Probably Notch signaling in these uncommittted precursors may lead to enhanced survival, preserving multilineage potential. The role of Notch pathway during human adult erytropiesis has not been described. The aim of this study is to investigate the modulation of Notch activity during “in vitro” human erythropiesis. Human CD34+ from perpheral blood of normal adult subjects were differentiate “in vitro” for two weeks by the addiction of IL-3, SCF and Epo. This method of colture reproduces all stages of adult erythropoiesis. We analized the modulation of the expression of Notch-1, of its effector Hes-1 and of several erythoid specific genes, at different stages of differentiation using real time PCR. Our analysis shows that Hes-1 expression, which indicates the activation of Notch-1 pathway, is very high in the early steps of differentiation (BFU-E, CFU-E) while in the late stages rapidly decreases to undetectable levels. The Notch-1 gene expression doesn’t seem to be modulated way, but we didn’t invstigate the protein levels yet. These data suggest that Notch pathway is involved in the early stages of erythroid differentiation where it may enhance erythroid progenitors survival up to CFU-E, as hypothisized in mouse model, preventing them from apoptotic stimuli and promoting their proliferation. Involvement of Notch-1 signaling in preventing erythroid progenitors from apoptosis during erythroid differentiation could be important in some erythropoietic disorders such as b-Thalassemia syndromes or diserythropoietic anemias.
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Cerisoli, Francesco, Letizia Cassinelli, Giuseppe Lamorte, Stefania Citterio, Maria Cristina Magli et Sergio Ottolenghi. « Efficient Expression of a Kit/GFP Transgene in Hematopoietic Stem Cells of Mouse Fetal Liver and Bone Marrow. » Blood 108, no 11 (16 novembre 2006) : 4159. http://dx.doi.org/10.1182/blood.v108.11.4159.4159.
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Abstract The hierarchy of transcription factors and signalling molecules involved in hematopoietic development has been dissected through transgenic and knock-out experiments, leading to the identification of several important genes. Less well known are the networks of transcription factors which regulate the activities of the main genes identified. Kit, encoding the membrane receptor of Stem Cell Factor (SCF), is a critical molecule for Hematopoietic Stem Cells (HSC) and some early progenitors, in which it is expressed. In a previous work (Cairns et al., Blood102, 3954;2003), we used mouse lines expressing transgenic Green Fluorescent Protein (GFP) under the control of Kit regulatory elements to investigate Kit regulation in different cell systems such as the hematopoietic and germ cell lineages. We generated a mouse Kit transgene capable of efficiently driving GFP expression both in PGC and in hematopoietic progenitors, such as CFU-Mix and BFU-Es. In the present work, we evaluated the functional efficiency of the same transgene also in HSC residing in the Fetal Liver (FL) and adult Bone Marrow (BM). To test if the construct is expressed in HSC, we transplanted FL or BM cells, fractionated on the basis of Kit expression and the level of GFP fluorescence, into irradiated non-transgenic mice. At the same time, the proportion of hematopoietic progenitors in the various fractions was assessed by in vitro colony assays. Following long term hematological reconstitution, the contribution of transplanted GFP cells was evaluated by the proportion of fluorescent mixed colonies in colture as well as by the proportion of fluorescent bone marrow cells, as assessed by FACS analysis. Long term reconstitution was confirmed by secondary transplants. Results show that the repopulating cells derived from fetal liver and adult bone marrow reside in a fraction of Kit+ cells with intermediate GFP fluorescence level, whereas CFU-Mix and BFU-E are in the highly GFP fluorescent fraction. Furthermore, flow cytometry of fetal liver shows that the intermediate fluorescence fraction is highly enriched in Kit+, Sca1+, CD11b+ cells (the expected HSC immunophenotype), whereas the high fluorescence fraction contains mainly Kit+, Sca1−, CD11b− cells. Similarly, the HSC-enriched tip of the Side Population (SP) of adult bone marrow is highly enriched in Kit+, Sca1+ cells of intermediate GFP fluorescence, whereas the upper part of the SP is enriched in Kit+, Sca1− cells of high GFP fluorescence. Our results indicate that the transgene (and possibly the endogenous Kit gene as well) might be transcribed at relatively low levels in HSC versus other progenitors. Noteworthy, the same transgene is also highly expressed in PGC and in Cardiac Stem Cells (CSC) (Messina et al., Circ. Res. 95,911;2004) and in blastocyst inner mass grown in vitro, indicating that the most 5′ part of the intron (4kb), added to the otherwise inactive promoter might include sites regulating Kit expression in multiple stem cell types.
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Bacigalupo, Andrea, Marisa Valle, Marina Podesta’, Anna Pitto, Elena Zocchi, Antonio De Flora, Sarah Pozzi et al. « The Suppressive Effect of Bone Marrow Mesenchymal Stem Cells on T Cell Activation, Is Deficient in Patients with Severe Aplastic Anemia. » Blood 104, no 11 (16 novembre 2004) : 506. http://dx.doi.org/10.1182/blood.v104.11.506.506.
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Abstract Normal bone marrow derived mesenchymal stem cells (MSC) suppress T cell function, and may provide an immunoprotected environment for hemopoietic stem cells. In this study we compare the suppressive activity on T cell function of MSC from 23 patients with severe aplastic anemia (SAA) patients, at diagnosis (n=3), following immunosuppressive therapy (IS) (n=16) or after an allogeneic bone marrow transplant (BMT) (n=4), and normal individuals. As shown in Figure 1 increasing numbers of control MSC (25, 50 and 100x10^3 cells) produced a dose dependent suppression of PHA induced T cell proliferation; when MSC were derived from SAA patients, they exhibited significantly less suppressive activity (37% vs 6%, 81% vs 24%, 96% vs 35%). The reduced ability of SAA MSC to suppress mitogen induced T cell proliferation, was seen irrespective of disease phase. Paired experiments on mixed lymphocyte reaction confirmed the inability of SAA MSC to suppress T cell function. Other abnormalities of SAA MSC included: (a) impaired capacity to down regulate CD38 expression on PHA primed T cells, (b) impaired ability to suppress gamma-IFN production in PHA coltures, which resulted in a 11 fold different gamma-IFN concentration in the cultures; (c) no effect on T cell mediated inhibition of hemopietic colony formation. Finally SAA MSC produced significantly (1 log) less adenosin diphosphate -ribosyl cyclase (cADPR), a promoter of in vitro hematopoiesis. MSC mediated suppression of PHA induced T cell proliferation, was restored to control levels in 3 of 4 patients post-BMT. In conclusion, the ability of MSC to down regulate T cell priming, proliferation and cytokine release is deficient in patients with SAA, persists indefinitively after immunosuppressive therapy, but may be restored after BMT. Whether this defect plays a role in the pathogenesis of marrow failure, remains to be determined. Figure Figure
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Catani, Lucia, Daria Sollazzo, Antonio Curti, Sara Trabanelli, Francesca Palandri, Nicola Polverelli, Michele Baccarani, Nicola Vianelli et Roberto Lemoli. « Impaired Interaction Between Regulatory T Cells and Dendritic Cells in Immune Thrombocytopenia. » Blood 114, no 22 (20 novembre 2009) : 3511. http://dx.doi.org/10.1182/blood.v114.22.3511.3511.
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Abstract Abstract 3511 Poster Board III-448 CD4+CD25+ regulatory T cells (Tregs) are critical in maintaining self-tolerance and preventing organ-specific autoimmune diseases. However, the role of Tregs in the pathogenesis of immune thrombocytopenia (ITP), an immune disorder in which increased platelet clearance is caused by antiplatelet autoantibodies, has not yet been clarified. The purpose of the present study was to investigate whether the interaction between dendritic cells (DCs) and regulatory T cells (Tregs) may play a pathogenetic role in ITP. Forty patients with active disease and 35 healthy subjects were enrolled into the study. We firstly characterized the number (by flow cytometry) and the suppressive activity (by modified allogeneic mixed leukocyte reaction) of Tregs in ITP. We found that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low-negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). We documented also that in ITP suppressive activity of Tregs was defective as compared to healthy subjects. In parallel experiments we studied the in vitro conversion of CD4+CD25- T cells, either from ITP patients or healthy subjects, into CD4+CD25+Foxp3+ Tregs by co-coltures with mature autologous/allogeneic DCs. The flow cytometry analysis showed that in ITP the low number of circulating Tregs may be partly due to the reduced ability of DCs to convert non-Treg cells into Tregs. We then explored the in vitro capability of CD4+CD25+ Tregs, either from ITP patients or healthy subjects, to inhibit allogeneic DCs maturation by flow cytometry evaluation of the expression of the costimulatory molecules CD80 and CD86. We demonstrated that in ITP patients CD4+CD25+ Tregs show lower ability to inhibit DCs maturation because they do not affect the expression of CD80 and CD86 molecules. This finding may be related to the lower level of Interleukin-10 and Interleukin-6 in the cocoltures. Taken together, these findings document that the interaction between DCs and Tregs is altered in ITP and suggest that this dysfunction may play a pathogenetic role. Supported in part by BolognaAIL (Italian association against Leukemia, Bologna section) Disclosures: No relevant conflicts of interest to declare.
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Piazza, Francesco A., Carmela Gurrieri, Gino Chioetto, Anna Colpo, Luca Bonanni, Barbara Montini, Laura Quotti Tubi et al. « Role of GSK3 in Multiple Myeloma Cell Growth and Survival. » Blood 110, no 11 (16 novembre 2007) : 2509. http://dx.doi.org/10.1182/blood.v110.11.2509.2509.
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Abstract The serine-threonine GSK3 displays a crucial role in different cancer-pathogenetic pathways, including the PI3K/AKT, Wnt β-catenin and NF-κB signaling cascades, either promoting or counteracting cell survival. The aim of this study was to investigate the role of GSK3 in multiple myeloma (MM) cell growth. GSK3α and β total and phosphorylated protein levels were found differentially expressed in malignant plasma cells as compared to normal resting B-lymphocytes and to normal in vitro generated plasmablasts. Intriguingly, in freshly isolated malignant plasmacells from patients, most of GSK3 was found colocalized with Wnt receptor LRP6 and casein kinase I next to the cell membrane as compared to normal plasmacells or B-cells from other malignancies, wher it was distributed in the cytosol and in the nucleus, thus suggesting a peculiar role of this kinase in these cells. Upon stimulation of MM cells with IL-6 and IGF-I, GSK3 enzymatic activity was hampered, while stimulation with TNFα did not affect GSK3 function nor the early events in NF-κB activation. Basal and IL-6 and IGF-I driven proliferation of MM cells was slightly impaired by GSK3 blockade. Interestingly, GSK3β−/− mouse embryo fibroblasts (MEFs) proliferated slightly slower as compared to GSK3β+/+ cells; however, GSK3α inhibition and IL-6 and IGF-I stimulation, resulted in much higher proliferation of GSK3β −/− cells. Intriguingly, GSK3 inhibition with specific compounds (SB216763 and SB415286) caused a significant rescue from cell death of growth factor-deprived MM cells while resulted in reduced cell proliferation and apoptosis of MM cells grown with serum or growth factors. When GSK3 inhibitors were added to MM cell cultured with bone marrow stromal cells (BMSC), MM cells survival increased and NF-κB and β-catenin-mediated gene transcription (of IAPs and cyclinD1, respectively) was deregulated. GSK3 activity inhibition did not modify the rate of proteasome inhibitor-induced cell death in co-colture experiments with BMSC, suggesting that the sensitivity to bortezomib-induced MM cell apoptosis is independent on GSK3. Altogether, our data indicate thatGSK3 localizes on the cell membrane in primary MM cells;GSK3 is differently regulated downstream from growth factors or TNFα-induced signaling pathways in MM cells;a peculiar role of GSK3 in malignant MM cells as compared to normal MEFs with regard to cell proliferation; anda critical role of this kinase in regulating the MM microenvironment.
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Burchielli, Emanuela, Antonella Tosti, Loredana Ruggeri, Katia Perruccio, Claudia De Angelis et Andrea Velardi. « Adoptive Therapy with T-Cell Precursors for Immune Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation. » Blood 114, no 22 (20 novembre 2009) : 3525. http://dx.doi.org/10.1182/blood.v114.22.3525.3525.
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Abstract Abstract 3525 Poster Board III-462 Recipients of allogeneic hematopoietic transplantation experience a slow reconstitution of donor-derived B and T cell number and function. This post-transplant period of immunodeficiency is associated with an increased risk of infection and malignant relapse. The developement of these complications notably correlates with the recovery of CD4+T cell subset. We proposed a strategy to enhances in vivo reconstitution by promoting donor-derived T cell development in the recipient's thymus. Recently Notch1-based ex-vivo system have been established to mature cord blood- or bone marrow-derived human HSCs into committed T-cell precursors. We used this system for the generation of T-cell precursors starting from G-CSF mobilized human HSCs. We cultured mobilized human CD34+ hematopoietic stem cells (HSCs) (2.5 × 105) in vitro on OP9 mouse stromal cells expressing the Notch 1 ligand Delta-like-1 (OP9-DL1) in the presence of rhFLT3-ligand (5ng/ml) and rhIL7 (5 ng/ml). After 6 weeks of co-culture we obtained a 3 log increase of human T-linage precursors of CD45RA+CD7high phenotype. Further co-colture (7-9 weeks) leed to the generation of CD4+ and CD8+ double-positive (DP) T cells and even mature CD4+ and CD8+ single positive (SP) ab-TCR lymphocytes. Experiments were designed in order to evaluate whether human CD45RA+CD7high T cell precursors could 1) engraft into NOD-SCID IL2 rg-/− mice 2) leed to in vivo expansion and maturation along T cell developmental pathway. Control mice were irradiated and transplanted with G-CSF-mobilized human CD34+ (dose 5×106 i.v.). 4 weeks after transplant more than 20% human CD45 positive cells engrafted in the bone marrow. Thymic engraftment occured at 8 weeks after transplant, with 80% human CD45 positive cells (thymic cellularity: 2.7×105 cells), mostly with T cell-immature phenotype of CD3-CD4-CD8 triple negative (95%) (TN) and CD4+CD8+double positive (5%) (DP). Co-transplant of CD45RA+CD7high T cell precursors (106 cells i.v.) along with CD34+HSC leed to an accelerated thymic engraftment (95% human CD45 positive cells; thymic cellularity 2.5 × 106 cells) already at 6 wks after transplant. Thymocytes were CD3-CD4-CD8 triple negative (51%) (TN) and CD4+CD8+double positive (DP) (42%) cells and at 8 weeks after transplant matured into CD3+CD4+ and CD3+CD8+ single positive (SP) T cells. Spectratyping analyses revealed a broad diversity of the T-cell receptor (TCR) repertoire. This occured in the complete absence of Graft versus Host Disease (GvHD) suggesting that adoptively transferred ex vivo-generated T-cell precursors developed into host-tolerant mature T cells. Ongonig experiment are needed to clarify the beneficial effect of adoptive immunotherapy with human T cell precursors on peripheral T cell reconstitution and control of infection in the humanized mouse system. We conclude that ex-vivo generation of human T-linage precursors is feasible from the G-CSF-mobilized HSCs and that can be succesfully tranfered in-vivo as a new strategie to enhance T-cell reconstitution after allogeneic HSCT with no risk of GvHD. Disclosures: No relevant conflicts of interest to declare.
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Chiarenza, Annalisa, Nunziatina Parrinello, Piera La Cava, Eleonora Spina, Daniele Tibullo, Cesarina Giallongo, Maide Cavalli et al. « Lenalidomide IS Able to Restore Immune SYSTEM IN MULTIPLE MYELOMA PATIENTS. » Blood 114, no 22 (20 novembre 2009) : 4909. http://dx.doi.org/10.1182/blood.v114.22.4909.4909.
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Abstract Abstract 4909 LENALIDOMIDE IS ABLE TO RESTORE IMMUNE SYSTEM IN MULTIPLE MYELOMA PATIENTS Annalisa Chiarenza, Nunziatina Parrinello, Piera La Cava, Eleonora Spina, Daniele Tibullo, Cesarina Giallongo, Maide Cavalli, Alessandra Romano, Paolo Fiumara, Giuseppe A. Palumbo, Francesco Di Raimondo Background Multiple myeloma (MM) is a malignant plasma-cell proliferative disorder associated with dysfunctional T-cell responses. The immunomodulatory Thal derivative (IMiD) CC-5013 (lenalidomide) appears to be a promising agent for the treatment of myeloma. Although the exact antitumor mechanism of action of lenalidomide is unknown, a number of mechanisms are postulated to be responsible for it's activity (inhibition of angiogenesis, direct antiproliferative and proapoptotic effects on MM cells, suppression of pro-inflammatory cytokines, modulation of myeloma-stromal cells adhesive interactions). In addition, it has been demonstrated that lenalidomide in vitro is able to enhance T cell proliferation and to promotes ADCC. In this study we evaluated if MM patients have a deficit of T-reg (CD4+, CD25+, and FOXP3+) and of T lymphocytes bearing CD200 (a tolerogenic molecule) and the effect of lenalidomide treatment on these parameters. In addition, we investigated whether lenalidomide could improve ex vivo the ADCC against myeloma cells. Materials and methods Eight patients with previously untreated MM (median age 56 years) were treated with lenalidomide plus dexamethasone as first line therapy. Lenalidomide was given orally 25 mg daily on days 1 to 21 of a 28-day cycle. Dexamethasone was given orally 40 mg daily on days 1, 8, 15, 22 of each cycle. All patients were evaluable for response and toxicity. Peripheral blood mononuclear cells (PBMNc) were obtained from MM patients using density gradient centrifugation (Fycoll) under sterile condictions, at the beginning of treatment and after 4 cycles of therapy. The percentage of T-reg (CD4+CD25+FOXP3+) and the expression of CD200 on T- lymphocytes were evaluated by cytometry. Twelve healthy subjects were used as control. Moreover, PBMNc (effector cells, E) were incubated with MM cells line ARH-77 (target cells, T), previously labelled with CFDA,SE (carboxyfluorescein diacetate, succinimidyl ester) as a tracing fluorescent marker, in culture medium (RPMI-1640, 10%FCS, 1%penicillin/streptomycin) at different concentration (T/E ratio 1:20, 1:40). After 18-24 h co-colture cells were analyzed by flow cytometry and MM plasma cells cytotoxicity was calculated as the percentage of positive CFDA,SE/propidium cells. Myeloma cell viability was determined by tripan blue esclusion and apoptosis was also evaluated using Annexin V/propidium assay. Two MM patients treated in first line with a combination of Velcade, Thalidomide and Dexamethasone (VTD) were used as control and the experiments were performed in duplicate. Results MM patients have a significantly lower rate of CD4+/CD25+/FOXP3+ and CD200+/CD3+ than normal (28,3±14,9/mmc and 37,8±24,7 /mmc vs 79,3±27,8 and 79,5± 48,9)(p=0,0001 and p=0,01 respectively). In our study, lenalidomide treatment resulted in an increase both of Treg cells and T-lymphocytes espressing CD200. This improvement is not statistically significant probably due to the low number of patients examined (tab I). More important, we observed that PBMC derived from patients treated with lenalidomide showed an increase ability to kill a target MM cell line compared to PBMC collected at diagnosis (CFDA,SE/propidium cells 11% vs 68%). This effect was more prominent in patients treated with lenalidomide than in MM patients treated with VTD (CFDA,SE/propidium cells 12% vs 39%), Fig.1. Conclusions Our data emphasize the role of lenalidomide in modulating the endogenous tumor-specific immune response and underline the anti-myeloma activity of these new class of drugs. Disclosures No relevant conflicts of interest to declare.
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Amodio, Nicola, Enrica Romeo, Maria Angelica Stamato, Eugenio Morelli, Mariateresa Fulciniti, Lavinia Raimondi, Marzia Leotta et al. « Selective Activation of the Non-Classical Estrogen Receptor Gper Elicits Potent Anti-Tumor Activity in Multiple Myeloma ». Blood 126, no 23 (3 décembre 2015) : 916. http://dx.doi.org/10.1182/blood.v126.23.916.916.
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Abstract Within the hematopoietic compartment, estrogens influence the differentiation, proliferation and survival of B cells and also increase the number of plasmacells (PCs) and their capacity to produce immunoglobulins. In addition to the classical estrogen receptors, an extensive body of literature has shed light on a 7-transmembrane spanning G-protein coupled estrogen receptor, GPER (also known as GPR30). The discovery of GPER-selective agonists and antagonists has led to the comprehension of the pathophysiological role of GPER and its involvement in cancer biology. However, the functional role of GPER in hematological malignancies has not been previously investigated. We detected GPER expression in 12 MM cell lines, including dexamethasone, bortezomib and carfilzomib-resistant cell lines, as well as in Waldestrom Macroglobulinemia (WM) cell lines either at mRNA and protein level, as assessed by qRT-PCR and western blotting, respectively. Importantly, MM patients' expression dataset analysis revealed a progressive decline of GPER mRNA levels during MM progression. Moreover, adhesion of MM cell lines (NCI-H929, U266) to bone marrow stromal cells (BMSCs) reduced GPER mRNA and protein levels even further, supporting a potential role of the BM microenvironment in regulating GPER expression. To address the role of GPER-mediated signaling on MM cell survival and cell death, first we tested the selective GPER agonist G-1 ((±)-1-[(3aR *,4S *,9bS *)-4-(6-Bromo-1,3-benzodioxol-5-yl) -3a,4,5,9b-tetrahydro-3H cyclopenta [c]quinolin-8-yl] ethanone) in vitro. G-1 inhibited, in a dose-dependent manner, the survival of WM (n=2) and MM cell lines (n=9), as well as of primary MM cells (n=3), with an IC50 at 48 h ranging from 2 to 3 μM, while it did not affect the viability of healthy donors' PBMCs (n=3); moreover, G-1 synergized with bortezomib in MM1S MM cells and with ibrutinib in BCMW1 WM cells. Both in MM and WM cell lines, G-1 treatment increased cells in G2/M phase and induced a potent and dose-dependent apoptotic cell death, as assessed by Annexin V/7AAD staining and western blot analysis of active caspases 3, 7 and 9. G-1-induced apoptosis was reverted by the pan-caspase inhibitor ZVAD-FMK and was not impaired by co-colture with BMSCs or by exogenous IL-6, IGF-1 or HGF. 17β-estradiol (10nM) and the selective GPER-antagonist G-15 (0.5µM) slightly increased the survival of MM and WM cells, and this effect was overcome by G-1 treatment. Moreover, G-1 induced the occurrence of a cytoprotective autophagy, as shown by the expression of autophagic markers, such as beclin-1 and LC3A/B, the cytosolic punctate pattern of LC3B and down-regulation of p62/SQSTM1 expression, and treatment of MM and WM cell lines with the autophagy inhibitor chloroquine potentiated G-1-triggered anti-survival effects. Importantly, intraperitoneal injection of G-1 (2mg/kg) in SCID mice significantly reduced the growth of subcutaneous MM1S and bortezomib-resistant AMO-abzb xenografts, and prolonged survival of treated animals. Since GPER-dependent molecular effects in solid tumors have been attributed to modulation of MAPK activity, by using an antibody array we analyzed the phosphorylation status of 24 protein kinases after GPER activation. Notably, treatment with G-1 (2µM) reduced the phosphorylation of ERK1/2, p38α-γ, AKT and GSK3α-β. Finally, on the light of the emerging role of miRNAs in MM pathobiology, we investigated by TaqMan qPCR the effects of G-1 on the miRNA profile and found that G-1 induced the expression of tumor suppressor miR-29b by down-regulating Sp1, the major miR-29b-negative regulator, thus disrupting the previously reported miR-29b/Sp1 negative feedback loop. Consistently, an inverse correlation between GPER and Sp1 mRNA levels emerged in MM patient PCs. In addition, miR-29b canonical targets, such as CDK6 and MCL-1, were downregulated in G-1-treated MM cells. Altogether, our results indicate that GPER is expressed in MM and WM and its selective activation via G-1 triggers potent anti-tumor activity through inhibition of oncogenic protein kinases and activation of miR-29b, providing the preclinical rationale for clinical investigation of GPER-agonists to treat these hematological malignancies. Disclosures No relevant conflicts of interest to declare.
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Civallero, Monica, Maria Cosenza, Samantha Pozzi et Stefano Sacchi. « Co-Targeting Jak Signaling Pathway and Histone Deacetylases Produces Synergistic Activity in Haematologic Malignancies Cell Lines ». Blood 128, no 22 (2 décembre 2016) : 4717. http://dx.doi.org/10.1182/blood.v128.22.4717.4717.
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Abstract Background and Objectives. Jak pathway is abnormally activated in multiple hematologic malignancies. Ruxolitinib, a JAK1/2 inhibitor, is currently prescribed in the clinical practice for the treatment of myelofibrosis; it is orally available and has a very manageable toxicity profile. Aberrant expression and activity of HDACs are also implicated in several types of tumors and HDAC inhibitors are under investigation in vitro and in clinical trials for the treatment of cancer. Vorinostat is a pan HDAC inhibitor with recognized efficacy in hematological disease, and ongoing studies are evaluating the best combination. Based on these considerations we screened a series of hematologic malignancies cell lines, in order to explore whether low doses of Ruxolitinib and Vorinostat might identify a new possible drug combination, with low toxicity profile. Methods. We tested Ruxolitinib and Vorinostat alone and in combination in 12 cell lines: Hodgkin Lymphoma (L1236, L540), B cell lymphomas (RPMI, WSU-NHL, Karpas422, RL) mantle cell lymphoma (Granta519, Jeko1), multiple myeloma (U266, RPMI8266), chronic limphocytic leukemia (MEK1), anaplastic lymphoma (Karpas299) and cutaneous T cell lymphoma (HUT78). The synergism was assessed by Chou-Talalay method. Effect of the treatment on cell cycle, apoptosis, caspase activation and ROS generation was evaluated by flow cytometry. Jak expression, apoptosis-regulating proteins and the phosphorylation status of protein kinases were studied by western blot analysis. Co-coltures with bone marrow stromal cells were also performed. Results. At 24h of treatment with ruxolitinib all the cell lines tested showed an homogeneous pattern of response, with IC50 around 20 nM; at 48h IC50 ranged from 0.1 to 2nM, with 4 cell lines more sensitive to the treatment (WSU-NHL, Karpas422, RL, HUT78) compared with the rest. The treatment with vorinostat induced an homogeneous response, both at 24h (IC50=40 uM) and 48h (IC50=5-10 uM). The combination treatment with a ratio R:V=0.1 nM:1 uM and 0.5nM:5uM showed a various range of results: a clear synergistic interaction of Ruxolitinib and Vorinostat was observed at 24h using low concentrations of two compounds in L1236, RL, RPMI, MEK1 and Karpas 299. There was no synergistic effects in mantle cell lymphoma cell lines, and a minor synergism in Karpas 422, a cell line that is known for carrying both t:(14;18) and t:(4;11) chromosomal translocation. The effect of the combination was additive on the remaining cell lines. The results were similar at 48 hours of treatment. The combination induced a marked increase in G2-M arrest and enhanced cell apoptosis in all cell lines. Accordingly the expression of apoptosis-regulating proteins, in particular BIM and Bad (SET-2), were up-regulated with down-regulation of Bcl-2 and Mcl-1 (HEL). Particularly evident was the effect of the combination on autophagy with decreased expression of p62 and a dramatic reactive oxygen species (ROS) accumulation in the cell lines in which the combination of the two drugs resulted highly synergistic. In addition, cell lines more sensitive to the combination of the two drugs showed a greater effect of the treatment on phosphorylation status of Jak, STAT3, STAT5, Akt and mTOR proteins. By co-culturing cells lines and primary BMSCs, we observed that Ruxolitinib and Vorinostat alone did not exert their anti-tumour activity in some cell lines. Conclusions. This study showed that the combination of Ruxolitinib and Vorinostat provokes significant changes in cell viability, apoptosis, cell cycle arrest, autophagy and ROS generation in several cell lines of hematologic malignancies alone and co-cultured. Because the bone microenvironment plays such an important role in the resistance to conventional therapies, the ability of Ruxolitinib combined with Vorinostat to overcome these factors is encouraging. Collectively, these findings create a compelling rationale to determine the in vivo activity of Ruxolitinib/Vorinostat combination. Disclosures No relevant conflicts of interest to declare.
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Todaro, Maria, Valentina Griggio, Candida Vitale, Chiara Salvetti, Chiara Riganti, Mario Boccadoro, Yosef Landesman et Marta Coscia. « Selinexor in Combination with Chemotherapy or Idelalisib Elicits a Synergistic Cytotoxic Effect in Primary CLL Cells, Also Overcoming Intrinsic and Stromal Cells-Mediated Fludarabine Resistance ». Blood 128, no 22 (2 décembre 2016) : 3210. http://dx.doi.org/10.1182/blood.v128.22.3210.3210.
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Abstract BACKGROUND: Despite the therapeutic efficacy of new target drugs in chronic lymphocytic leukemia (CLL), treatment of high-risk patients remains an unmet clinical need. Disease aggressiveness can be ascribed to intrinsic features of the tumor cells such as immunoglobulin heavy chain (IGHV) mutational status and TP53 disruption, which are strong determinant of drug response. Many of the tumor suppressor and growth regulatory proteins with a known pathogenic role in CLL (i.e. p53, NF-kB, Akt, IkB) bind the nuclear export protein XPO1 (Chromosome Region Maintenance 1; CRM1) and are carried through the nuclear pore complex into the cell cytoplasm. Elevated protein levels of XPO1 and specific XPO1 mutations have been reported in various hematologic and solid tumors. In particular, XPO1 is overexpressed and recurrently mutated in CLL cells (Puente XS et al, Nature 2011; Lapalombella R et al, Blood 2012). Selinexor (KPT-330) an oral inhibitor of XPO1, is active as single agent in different hematologic malignancies including acute myeloid leukemia, non-Hodgkin lymphomas, CLL and multiple myeloma. The combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival of a CLL mouse model compared with ibrutinib alone (Hing ZA et al, Blood 2015). AIM: The aim of this study is to evaluate the additive or synergistic in vitro cytotoxic effects of selinexor, used in combination with chemotherapeutic drugs or the new PI3k inhibitor idelalisib against primary CLL cells. Specifically, this study aims at identifying combination regimens that might overcome single agent resistance. METHODS: 15 patients with CLL were included in the study, among these 9 with mutated (M) and 3 patients with unmutated (UM) IGHV; for 3 patients the mutational status was not available at the moment of data analyses. Purified CLL cells were exposed, alone or in presence of the murine stromal cell line M2-10B4, to selinexor (10 nM, 100 nM, 1 uM and 10 uM) in combination with fludarabine (F-ara-A, 10 nM, 100 nM, 1 uM and 10 uM), bendamustine (Ben, 3 mM, 10 mM, 30 mM and 50 mM) or idelalisib (Ide, 10 nM, 100 nM, 1 uM and 10 uM) for 24, 72 and 120 hours. Cell viability was analysed by Annexin-V/propidium Iodide (AnnV/PI) immunostaining and flow cytometry. Samples were considered resistant when the relative viability of F-ara-A treated CLL cells compared to untreated control was >0.5. Combination analysis was performed using Calcusyn software; combinations were considered synergistic when CI was <1. RESULTS: Leukemic cells were cultured in the presence of increasing concentrations of selinexor, used alone or in combination with F-ara-A and Ben, and with the PI3Kδ inhibitor, Ide. After 72 hours of culture, the mean percentage of viable AnnV-/PI- CLL cells significantly decreased by 0,62-ratio following KPT-330 (100 nM), 0,42-ratio following F-ara-A (1 uM) and 0,24-ratio after dual treatment, compared to untreated controls. Combination analysis showed that selinexor and F-ara-A strongly synergize in inducing CLL cells apoptosis with a CI < 1. Similarly, we observed a synergistic interaction between selinexor (100 nM) and Ben (30 mM) that significantly enhanced the cytotoxic effect of the individual drugs (0,62-ratio for selinexor, 0,68-ratio for Ben and 0,41-ratio for selinexor + Ben) with a CI <1, at the same time point. The combination between selinexor (100 nM) and Ide (10 nM) at 72 hours resulted in a weaker, although significant, viability reduction (0,62-ratio for KPT-330, 0,5 for Ide and 0,38 for selinexor + Ide). We observed that IGHV UM CLL cells showed higher fold reduction values in cell viability when exposed to synergistic combinations, compared to IGHV M cells. Selinexor (100 nM) was also effective in impairing the viability of CLL cells that showed intrinsic resistance to F-ara-A (n=6, 0,61-ratio for selinexor, 0,74-ratio for F-ara-A and 0,4-ratio for selinexor + F-ara-A). Lastly, we exposed CLL-stromal cells co-coltures to the identified synergistic combinations and found that selinexor + F-ara-A or selinexor + Ide significantly reduced the viability of leukemic cells, effectively counteracting the protective effect exerted by stromal cells toward drug-induced apoptosis. CONCLUSIONS: Our data demonstrate that the combination of selinexor with chemotherapy or Ide has synergistic cytotoxic effects, also counteracting intrinsic or stromal cells-mediated drug resistance. Disclosures Boccadoro: SANOFI: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Abbivie: Honoraria; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Research Funding. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Coscia:ROCHE: Honoraria, Other: Advisory board; Karyopharm: Research Funding; Mundipharma: Honoraria; Janssen: Honoraria; Gilead: Honoraria.
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Ruta, Claudia, et Maurizio Lambardi. « La coltura in vitro per la conservazione della biodiversità orticola ». Italus Hortus, no 25 (2018). http://dx.doi.org/10.26353/j.itahort/2018.1.3960.
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« Società Botanica Italiana : Gruppo Di Lavoro Su Differenziamento E Coltura Di Tessuti : Riunione Scientifica Su Aspetti Di Ricerca Di Base E Biotecnologie Della Coltura Di Tessuti Vegetal1 In Vitro : S. Miniato, 5-6 giugno 1987 ». Giornale botanico italiano 123, no 1-2 (janvier 1989) : 109–38. http://dx.doi.org/10.1080/11263508909430250.
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Suaudeau, Jacques. « Le cellule staminali : dall’applicazione clinica al parere etico Parte I. Le cellule staminali embrionali ». Medicina e Morale 55, no 4 (30 août 2006). http://dx.doi.org/10.4081/mem.2006.346.
Texte intégralRésumé :
Otto anni dopo l'inizio della ricerca sulle cellule staminali umane, sembra essere arrivato il momento di considerare oggettivamente quale possa essere il futuro di tale ricerca, e quali siano i problemi etici collegati. In questo articolo sono considerate le cellule staminali embrionali (ES) a livello tecnico e clinico. L'interesse particolare di tali cellule risiede nella loro capacità di continua proliferazione indifferenziata e di stabile sviluppo potenziale in un’ampia tipologia di cellule, anche dopo una coltura prolungata. Numerosi lavori mostrano, in particolare, che le cellule ES possono essere differenziate in neuroni e glia ed integrarsi nel tessuto neurale in animali riceventi. La differenziazione verso neuroni dopaminergici è stata ottenuta per le cellule staminali embrionali umane (hES) con promesse per il trattamento clinico della malattia di Parkinson. Le cellule ES hanno anche dimostrato la capacità di facilitare il recupero del danno del midollo spinale, nel topo. L'innesto di cellule ES in ratti con infarto miocardico provoca un miglioramento a lungo termine della funzione del cuore ed aumenta la percentuale di sopravvivenza. Tuttavia, ci sono molti ostacoli che devono essere superati prima di pensare ad un uso clinico di tali cellule. Il problema forse più complesso è di poter dirigere in modo efficiente e riproducibile la differenziazione delle cellule ES attraverso percorsi specifici. In secondo luogo, il rischio di difetti o instabilità epigenetiche nelle cellule ES è reale, tenendo conto della loro origine da embrioni ottenuti da fecondazione in vitro e del processo di coltura di tali cellule, una volta individuate. Terzo, le cellule ES allo stato indifferenziato sono cancerogeniche, il che, per un uso clinico, rende necessaria la loro differenziazione e l’attenta eliminazione di cellule ES rimaste indifferenziate. Infine, l'uso clinico delle cellule ES richiede la soluzione del problema immunologico della compatibilità HLA con il ricevente. A tale scopo sono state proposte varie soluzioni, per prima il trasferimento nucleare, detto anche “clonazione terapeutica”. Allo stato attuale essa non è applicabile ai primati ed alla specie umana. Inoltre sarebbe necessaria una quantità enorme ed irrealistica di ovociti umani. Ci si orienta oggi, anche per motivi etici, verso soluzioni "alternative" come il trasferimento nucleare modificato, nel quale si producono embrioni deficitari incapaci di svilupparsi correttamente, la partenogenesi, la raccolta di blastomeri in occasione della diagnosi preimpiantatoria, o la riprogrammazione delle cellule staminali somatiche. Ad oggi, lo studio delle cellule staminali embrionali rappresenta una promettente chiave per futuri progressi in ambito biologico (biologia dello sviluppo, biologia cellulare e biologia molecolare), nella misura in cui permette di capire meglio i processi ed i meccanismi della differenziazione e della rigenerazione dei tessuti. ---------- Eight years after the onset of the investigation on embryonic stem cells (ESCs), it seems that time has come to consider objectively what the future of such research can be, and what are the ethical issues that are involved. In this first part ESCs are considered at the technical and clinical level. The particular interest of such cells resides in their ability for endless undifferentiated proliferation and for potential development in a large array of various types of cells, even after prolonged culture. A large amount of studies show in particular that ESCs can differentiate in neurons and glia and integrate in the neural tissue of recipient animals. The promotion of such differentiation toward dopaminergic neurons has been obtained for human embryonic stem cells (hESCS), which is promising for possible future clinical application to the treatment of Parkinson's disease. The ESCs have also demonstrated their ability to facilitate the recovery of damaged spinal cord in mice. The graft of ESCs in the hearts of rats with myocardial infarction leads to an improvement of heart function and increases survival. Nevertheless, there are many obstacles that must be overcome before thinking to a clinical use of such cells. The problem perhaps more complex is to be able to direct in an efficient and reproducible way the differentiation of the ESCs in culture. Second, the risk of epigenetic defects or instability with ESCs is real, keeping in mind their origin from embryos created by in vitro fertilization, and the fact that they are kept proliferating in culture for a long period of time, once individualized. Third, ESCs in the undifferentiated state generate cancers when injected in tissues, and that makes necessary, for a clinical use, to start their differentiation in vitro and then to eliminate carefully from the end product these ESCs that are still undifferentiated. Finally, the clinical use of ESCs supposes resolved the immunological problem of their HLA compatibility with the patient who will receive them. Various solutions have been proposed for resolving this last problem, with, in first line, nuclear transfer, the so called "therapeutic cloning." Up to now this nuclear transfer has not been successful in primates and humans. Moreover, it would require the availability of unrealistically large amounts of human ovocytes. Today, also for ethical reasons, the tendency is to look after "alternative solutions" such as "altered nuclear transfer", in which are created disabled embryos, unable to develop correctly, parthenogenesis, the harvest of human blastomeres in the course of preimplantation diagnosis or the reprogramming of human somatic stem cells to an "embryonic state". At present time, the study of ESCs represents a promising key to progresses in the knowledge of cellular and molecular aspects of development, healing and tissue regeneration. These progresses may in turn lead to clinical applications, especially in the field of degenerative diseases and for the recovery of damaged tissues and organs.
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FRANZIN, ROSSANA, Alessandra Stasi, Fabio Sallustio, Chiara Divella, Guido Merlotti, Marco Quaglia, Claudia Curci et al. « TO007PLASMA EXTRACELLULAR VESICLES MEDIATE ENDOTHELIAL TO MESENCHYMAL TRANSITION AND TUBULAR SENESCENCE IN RENAL ANTIBODY MEDIATED REJECTION BY COMPLEMENT ACTIVATION ». Nephrology Dialysis Transplantation 35, Supplement_3 (1 juin 2020). http://dx.doi.org/10.1093/ndt/gfaa141.to007.
Texte intégralRésumé :
Abstract Background and Aims EVs (Extracellular vesicles) are circulating microparticles able to mediate cell-to cell communication by carrying proteins, DNA, RNA or antibodies on their surface. EV are emerging as pivotal in renal Antibody-mediated rejection (AMR), one of the major causes of transplant failure associated to a massive complement activation. AMR is characterized by endothelial to mesenchymal transition (EndMT) and the occurrence of tubular accelerated senescence process, known as graft “Inflammaging”. However, the potential role of EVs in accelerating the renal aging and graft fibrosis after AMR is not well understood. Method Plasma EVs were isolated from 10 Acute AMR (AAMR), 10 Chronic AMR (CAMR) patients, 5 stable graft transplanted patients and 5 healthy volunteers. EVs were isolated by ultracentrifugation at 100.000 g for 1h at 4°C, quantified by Nanoparticle Tracking Analysis (Nanosight, NTA EV/ml) and characterized by FACS (Attune Nxt). RPTEC and endothelial cell culture were incubated with EVs (5e+4 EVs/cells target for 24h. MTT test was performed to assess cell viability. Cellular senescence was investigated by qPCR for p21, p53, Klotho and CYP1B1 and SA-β-gal staining were performed. To assess EndMT analysis for CD31, VE-cadherin, Vimentin, Collagen I was performed by FACS. mRNA level of C3 and CFH were also measured by qPCR and C4d deposits were evaluated in endothelial cell colture by IF. Renal biopsies were then analyzed for inflammaging (p16INK4a) and EndMT markers (CD31/αSMA) by IHC and IF. Results The Nanosight analysis showed significant differences in the amount of-EVs count per ml of plasma in AMR patients compared to healthy subjects. EVs appeared to be significantly augmented in acute AMR, in a higher manner than chronic AMR (NTA, p=0.0154). By cytofluorimetric analysis, the endothelial markers CD31 and VE- cadherin appeared to be significantly increased compared of healthy control, indicating a predominant endothelial EV origin (p<0.05). Furthermore, an increase for CD3, CD4, CD8, CD40 and CD40L lymphocyte/monocytes markers was found. In vitro, the exposure of RPTEC and endothelial cells to AMR-derived EVs did not induce the loss of cell proliferation. However, AMR-derived EVs induced senescence in RPTEC, as observed by increase in SA-β-gal positive cells, upregulation of p21, p53, CYP1B1 and downregulated KL gene expression (p<0.05). EVs induced the EndMT as observed by FACS with the downregulation of CD31, VE-cadherin (CD144) and increase of Vimentin and Collagen I (p=0.025). To evaluate the contribution of EVs to local complement activation, C3 and CFH qPCR analysis were performed. EVs from AMR patients induced a significant increase in C3 gene expression with concomitant downregulation in CFH in RPTEC. EV exposition induced the classical and lectin pathway activation in endothelial culture medium, and C4d deposition. These data support the hypothesis that circulating EVs can amplify local complement activation in systemic endothelial and tubular cells during AMR, therefore leading to accelerated senescence and fibrosis as later effects. Finally, renal AMR biopsies showed significant tubular senescence as indicated by p16 expression; p16 was significantly upregulated in Chronic compared with Acute AMR biopsies (p<0.05). The AMR biopsies showed positivity for EndMT, as indicated by CD31 decrease and interstitial αSMA upregulation (p<0.05). Conclusion These results suggest a putative role for circulating AMR derived-EVs in inducing the tubular inflammaging by local complement activation and early fibrosis by EndMT. The EVs cargo characterization that is ongoing might highlight novel targets for therapeutic intervention.