1,720,974 research outputs found
MicroRNA e il recettore adenosinico A3 come biomarcatori del mesotelioma maligno della pleura
No full textBackground: Malignant pleural mesothelioma (MPM) is an aggressive asbestosrelated
cancer that develops via mesothelial cell transformation. At present there
are no effective therapies for MPM. Great efforts have been made in finding
specific markers/mechanisms for MPM onset, including studies into microRNAs
and adenosine receptors (ARs). Recent studies have shown the differential
expression of mature microRNAs in several human cancers, suggesting their
potential role as oncogenes or tumor suppressor genes, and the involvement of
ARs in the regulation of cell death and proliferation.
Methods: In this study, we investigated miRNAs profile and the expression of
A3ARs in MPM. MiRNAs profile was investigated in 5 HMCs (human normal
pleural mesothelial short term cell cultures) and 5 MPMs, with microarray
approach. These results were confirmed by Real Time quantitative RT-PCR and
western blotting. ARs were analyzed by using RT-PCR, western blotting and
saturation binding assays. HMC were treated with crocidolite asbestos which is the
principal risk factor of MM. The role of A3ARs on these cellular models,
evaluating cAMP production, Akt phosphorylation and NF-kB activation was
investigated. The dual effect of A3AR stimulation on healthy and cancer cell
growth was studied by means of proliferation, apoptosis and cytotoxicity assays.
Results: A comparative analysis of miRNA expression in MPM and HMCs was
carried out. Microarray profiling showed different miRNA expression between
MPM and HMCs. Specifically, 13 miRNAs (17-5p, 18a, 19b, 20a, 20b, 25, 92,
106a, 106b), members of the oncomiRNA miR 17-92 and its paralogs were markedly dysregulated. Besides, in our investigation, additional miRNAs, such as
miR-7, miR-182, miR-214 and miR-497 were found to be dysregulated in MPM.
A3AR was up-regulated by 2.5 fold (P<0.01) in MMP when compared with HMP.
Stimulation of A3ARs decreases proliferation and exerts cytotoxic and proapoptotic
effect on MMC and on HMC exposed to asbestos and TNF-�, but not in
HMC with an involvement of the de-regulation of Akt/NF-kB cell survival
pathway.
Conclusion: These data are in agreement with results which have previously been
reported on dysregulated miRNAs for other solid human tumors. Moreover, in our
investigation, additional miRNAs were found to be dysregulated in MPM.
Interestingly, gene products which regulate the cell cycle are targets and predicted
targets for these miRNAs. Our data suggest that specific miRNAs and A3ARs
could be key players in MPM development/progression. In addition, some of these
miRNAs may represent MPM markers and potential targets for new therapeutic
approaches. Besides, our data suggest that A3AR could represent a
pharmacological target to prevent tumor development after asbestos exposure and
to treat full blown MPM
ALLESTIMENTO DI UN NUOVO SAGGIO ELISA INDIRETTO PER RIVELARE ANTICORPI SPECIFICI CONTRO L’ONCOPROTEINA ANTIGENE T GRANDE DI SV40 NEI SIERI DI INDIVIDUI SANI E PAZIENTI ONCOLOGICI.
No full textIl virus oncogeno della scimmia SV40 appartiene ai virus Polioma. SV40 è endemico nei macachi asiatici ed è stato accidentalmente somministrato all’uomo con i vaccini antipolio contaminati da questo virus nel periodo 1955-1963. In anni recenti, sequenze genomiche di SV40 e la sua oncoproteina antigene T (agT) sono state rilevate in diverse neoplasie umane, come tumori cerebrali, mesoteliomi, linfomi ed osteosarcomi. Inoltre, SV40 è stato trovato associato al sangue periferico, sperma e urina di individui sani [1, 2]
Campioni di siero di pazienti oncologici- e di individui sani, suddivisi per età e sesso, sono stati valutati con un test ELISA sperimentale per la ricerca di anticorpi anti-antigene T di SV40. Gli antigeni di SV40, rappresentati da peptici sintetici, denominati peptidi x, y, z, sono stati selezionati tra i non cross-reattivi con altri antigeni di virus Polioma omologhi, come BK e JC, ubiquitari nella popolazione umana. Successivamente, i corrispondenti buffy coat di pazienti e individui sani, scelti tra i campioni SV40-positivi in ELISA con densità ottica elevata, o nettamente SV40-negativi, sono stati sottoposti ad analisi di PCR per la ricerca di sequenze genomiche di SV40.
I campioni di siero hanno mostrato la presenza di anticorpi anti-antigene T di SV40. La SV40-positività e la SV40-negatività in ELISA è stata confermata dalla analisi di PCR.
I nostri dati indicano che SV40 circola nella popolazione umana. Tuttavia la sua prevalenza appare limitata rispetto a quella dei virus Polioma umani BK e JC. La conferma della presenza di SV40 nella popolazione umana apre nuove prospettive di studio per comprenderne le vie di trasmissione e il suo potenziale coinvolgimento in patologie sia neoplastiche che non-neoplastiche
Mesenchymal stem cell behavior in biomaterials assays
No full textIntroduction: in the orthopedic perspective, tissue engineering is focused on the development of innovative materials, whose action consists in recruiting bone progenitor cells and in stimulating their proliferation. In this study, we investigated the interaction between human mesenchymal stem cells from adults and two types of biomaterials, which differ for chemical composition and format. To evaluate the effects of porosity on cell adhesion and proliferation comparative analyses were carried out on different forms of two bioceramic materials.
Materials and Methods: Bone marrow aspirates from iliac crests were obtained from orthopedic patients who underwent bone marrow harvesting under general anesthesia. Mesenchymal stem cells (hMSCs) were isolated by Ficoll-mediated discontinuous density gradient centrifugation and polystyrene adherence capacity. Cell cultures at the second passage were characterized, by the flow cytometric analysis (FCA) of specific surface antigens. A surface marker-expression pattern, positive (Stro-1, CD29, CD44, CD71, CD73, CD90, CD105) and negative (CD45, CD34, CD235), was employed to characterize the cells obtained. hMSCs were expanded and duplication time evaluated. The AlamarBlue assay was used to evaluate the number of cells attached to the biomaterials and to the control in adhesion and proliferation assay. The quantification of the relative amount of Focal Adhesion Kinase was employed to evaluate the strength of the cell adhesion to the biomaterials after 36 hours from seeding. In order to verify the morphology of the hMSCs attached to bioceramics, stem cells grown on the biomaterials were analyzed by SEM. Moreover, to verify whether the cytoskeletal organization of hMSCs cultured on the analyzed biomaterials was altered, actin fibers were investigated using TRITC conjugated-Phalloidin.
Results: the biomaterials assayed in our study provided a favorable environment for hMSCs adhesion and growth, without any evidence of cytotoxic effects. Indeed, hMSCs adhesion and growth assays indicate that shape and porosity influence the cytocompatibility of bioceramics. We may infer that intra- and iter- porosity of biomaterials investigated herein, are determinant factors influencing hMSCs behavior. Besides, the correlation between vitality and a greater amount of Focal Adhesion Kinase at molecular level show how cellular processes may be modulated by structured surface and three dimensional architecture of biomaterials. hMSC SEM analysis showed an elongated spindle-like morphology. The cells adhered to biomaterials with many dendritic projections with some differences in the morphology due to scaffolds structure. Moreover, hMSCs cytoskeletal architecture on biomaterials was indistinguishable from that of hMSCs grown on plastic petri dishes, used as a control.
Conclusion: These assays confirm the feasibility to evaluate biomaterials biological features in pre-clinical environment with patients mesenchymal stem cells in order to obtain information about subject specific response. Moreover, the development of new matrices have to consider the possibility to employ bioactive surfaces able to modulate cell adhesion and differentiation processes
Merkel Cell Polyomavirus (MCV) in Buffy Coats of Healthy Blood Donors.
No full textBackground.
Merkel cell polyomavirus (MCV), a DNA tumor virus, has been found to be associated with Merkel cell carcinoma (MCC) and chronic lymphocytic leukaemia (CLL). MCV sequences have also been detected in various normal tissues in tumor affected patients. Immunologic studies have detected MCV antibodies in as many as 80% of healthy blood donors. This high seroprevalence suggests that MCV infection is widespread in humans.
Materials and methods
DNA from buffy coats (n=60) of healthy blood donors was investigated by two PCR rounds, 35 cycles each, for two different MCV Tag regions, nt 571-879 and nt 1709-1846, respectively. To quantify the MCV DNA load, positive samples were further analyzed by RQ-PCR for Tag sequences. To determine the human cell equivalents of each sample under analysis, RQ-PCR assays were carried out simultaneously with the cellular RNase P gene. The specificity of PCR amplified products, 10 amplicons from the MCV Tag regions, were DNA sequenced.
Results
PCR re-amplifications showed that 13 out of 60 (22%) DNA samples were positive for MCV Tag coding sequences. Buffy coats under analysis indicated that the viral DNA copy numbers were very low, ranging from 10 to 100 copies/100,000 cells. The sequencing result of the PCR amplicons, for both strands, nt 571-879 and nt 1709-nt 1846, identified as belonging to the MCV genome, MKL-1 strain.
Conclusions
MCV sequences were detected in buffy coats from healthy blood donors. This result suggests that MCV is able to infect specific blood leukocyte cells, where it remains in a latent/persistent state in the PBMCs of immune-competent individuals. In the long term, viral persistent infection may allow MCV to generate mutants which can participate in the cell transformation process. Indeed, large T antigen MCV deletion mutants have been detected in CLL or integrated into MCC. This oncogenic process, together with the immune impairment of the host and other factors, is a well-known multistep cell transformation mechanism employed by other DNA tumor viruses, such as human papillomaviruses, which are closely related to the Merkel cell polyomavirus.
Acknowledgements
We thank Prof. Tobias Allander, Karolinska Instituten, Stockolm, Sweden, for his generous gift of the recombinant plasmid pMCV-LT.1
Reference
Pancaldi C, Corazzari V, Maniero S, Mazzoni E, Comar M, Martini F, Tognon M. Merkel cell polyomavirus DNA sequences in the buffy coats of healthy blood donors. Blood 2011, 117:7099-7101
ANALISI DI PCR QUALITATIVA E QUANTITATIVA DEL DNA DEL VIRUS ONCOGENO SV40 IN CAMPIONI DI SANGUE DI DONATORI DI CASALE MONFERRATO, CITTÀ CONTAMINATA DALL’AMIANTO
No full textL’amianto è considerato l’agente tumorale responsabile dell’insorgenza del mesotelioma maligno della pleura (MM). MM è un tumore molto aggressivo, attualmente privo di cura, la cui incidenza sta aumentando in molti Paesi, soprattutto nelle coorti di lavoratori ex esposti all’amianto. La predisposizione genetica e l’infezione da virus oncogeno SV40 sono considerati come altri fattori coinvolti nell’insorgenza e progressione del mesotelioma maligno della pleura,. SV40 è un potente virus oncogeno a DNA, accidentalmente somministrato alla popolazione umana su scala planetaria negli anni 1955-63 con i primi vaccini allestiti contro la poliomielite, sia di Salk che di Sabin. SV40 infetta naturalmente il macaco asiatico ed era presente nelle colture primarie di cellule di rene di scimmia impiegate per produrre i primi vaccini antipolio. Molti dati sperimentali indicano che SV40 è anche un virus umano. La trasmissione orizzontale dell’infezione sembra avvenire con diverse modalità, come per via sessuale, ematica, oro-fecale e respiratoria e con le urine contaminate. In condizioni sperimentali SV40 trasforma differenti linee cellulari animali e umane, mentre inoculato per diverse vie in animali da laboratorio induce specifici tumori, come tumori cerebrali, mesoteliomi, osteosarcomi e linfomi. Il potenziale trasformante ed oncogeno di SV40 risiede nella sua principale oncoproteina, l’antigene T grande (agT). Sequenze del DNA di SV40 e l’espressione dell’agT sono state evidenziate in specifici tumori umani, dello stesso tipo indotti negli animali. Ad esempio sequenze di SV40 e l’espressione dell’agT sono state mostrate in circa il 50% dei mesoteliomi maligni della pleura.
Scopo di questo studio è stato di verificare la presenza di sequenze del DNA di SV40 in cellule del sangue periferico di donatori sani provenienti dal Centro Trasfusionale di Casale Monferrato. In questa cittadina industriale, pesantemente contaminata dall’amianto, l’incidenza del mesotelioma maligno della pleura nella popolazione è di dieci volte superiore rispetto ad aree non contaminate con l’amianto. Il DNA estratto da buffy coats, raccolti presso il locale Centro Trasfusionale, è stato analizzato con saggi di PCR qualitativa e quantitativa.
La presenza di sequenze codificanti per la regione ammino-terminale dell’agT di SV40 è stata indagata mediante l’analisi di nested PCR. Tali sequenze specifiche sono state evidenziate in 24/148 (16%) campioni. In seguito, i campioni positivi per l’agT sono stati analizzati per le sequenze della regione di regolazione di SV40. L’analisi di nested PCR ha mostrato che 17/24 campioni sono positivi anche per queste sequenze virali. E’ interessante notare che la regione di regolazione del DNA di SV40 contiene sequenze specifiche che consentono di distinguere tra loro i tre principali virus polioma (SV40, BK e JC) e i diversi ceppi di SV40. Nove amplificati della regione di regolazione di SV40, di campioni diversi di buffy coats, sono stati sottoposti ad analisi di sequenziamento del DNA. La sequenza nucleotidica indica che i nove amplificati appartengono al ceppo selvatico 776.
Allo scopo di quantificare nei campioni di sangue SV40-positivi la carica del DNA virale sono state eseguiti saggi di Real-Time PCR su 22 DNA estratti da buffy coats. Questa analisi ha confermato che i 22 campioni erano SV40-positivi per le sequenze dell’agT, con un numero di copie di DNA virale comprese tra 10 e 104 per 100,000 cellule o genomi equivalenti.
La nostra ricerca indica che la prevalenza di SV40 in campioni di buffy coats di donatori sani di Casale Monferrato, è simile a quella riscontrata in altri donatori di sangue di aree non contaminate dall’amianto. I nostri risultati indicano che l’amianto presente nell’ambiente di Casal Monferrato non facilita la diffusione dell’infezione di SV40, almeno tra i donatori sani analizzati. Tuttavia, la presenza di sequenze di SV40 nei donatori suggerisce l’ipotesi che amianto e virus oncogeno agiscono come co-fattori nell’insorgenza dell’MM. Infatti, il minerale amianto oltre ad essere agente tumorale è anche un potente immunodeppressivo che potrebbe favorire la diffusione dell’infezione di SV40 nei tessuti dell’ospite. In condizioni peculiari dell’individuo/paziente si potrebbe quindi innescare l’azione combinata tra amianto e virus oncogeno durante il meccanismo multifase della tumorigenesi che nei lavoratori ex esposti all’amianto può durare decenni.
I nostri risultati relativi alla presenza di sequenze di SV40 nei donatori di sangue indicano che il virus oncogeno SV40 circola nella popolazione umana. La sua prevalenza è di circa il 15%-20%, mentre il numero di copie di DNA virale nei campioni positivi risulta essere di bassa entità
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Extended lifespan of normal human B lymphocytes experimentally infected by SV40 or transfected by SV40 large T antigen expression vector
No full textSV40 footprints were detected in different lymphoproliferative disorders and in blood specimens of healthy donors. However, little is known on the ability of SV40 to infect/transform normal human B-lymphocytes. In this in vitro study, experimental SV40 infection and SV40 Tag transfection of normal human B-lymphocytes from healthy blood donors were carried out. In SV40 infected /transfected purified B-cells, during the time course analyses, viral DNA sequences were detected by PCR, while Tag mRNA and protein were revealed by RT-PCR and immunocytochemistry, respectively. Trypan blue and Alamar blue assays showed an increase in number of cells and cell viability of infected/transfected B-cells up to day 50, then a drastic and constant cell number reduction was observed in cultures. Approximately 50% of both infected and transfected B-cells appeared morphologically transformed. SV40 viral progeny and its titer from infected B-cells was determined by plaque assay in permissive CV-1 cells. Our data indicate that human B-cells can be efficiently infected by SV40, release a viral progeny, while at the same time are transformed. SV40 infected/Tag transfected B-cells may represent an experimental model of study for investigating new biomarkers and targets for innovative therapeutic approaches in human B-cell malignancies
ASSOCIAZIONE TRA I LINFOMI NON-HODGKIN E IL VIRUS ONCOGENO SV40 .
No full textSV40 (Simian Virus 40) è un virus tumorale a DNA. Sequenze genomiche di SV40 e l’espressione della sua oncoproteina antigene T grande (agT) sono state rilevate in alcuni tumori umani quali osteosarcomi, mesoteliomi, tumori cerebrali e linfomi non-Hodgkin. In questo studio abbiamo indagato l’associazione tra i linfomi non-Hodgkin e SV40. In una prima fase abbiamo ricercato la presenza di anticorpi specifici contro SV40 nel siero di pazienti affetti da linfomi non-Hodgkin. L’indagine è stata eseguita con un saggio ELISA indiretto impiegando specifici peptidi sintetici corrispondenti sia alla regione precoce che a quella tardiva. Nei sieri di pazienti affetti da linfomi non-Hodgkin la prevalenza di anticorpi contro le proteine capsidiche del virus è risultata del 46%, mentre quella verso l’oncoproteina virale agT è stata del 51%. Nella coorte di controllo, rappresentata da donatori di sangue le prevalenze di anticorpi contro le stesse proteine sono del 18% e 19%, rispettivamente. L’analisi immunologica conferma che l’associazione tra SV40 e linfomi non-Hodgkin è statisticamente significativa..
In una seconda fase del lavoro abbiamo studiato, in un modello in vitro di colture cellulari primarie di linfociti, la capacità trasformante di SV40 nei confronti dei linfociti B e T umani. La capacità trasformante di SV40 è stata saggiata a diversi tempi di trasfezione del DNA virale. Analisi molecolari e saggi di vitalità rivelano che SV40 è in grado di trasformare sia i linfociti B che i linfociti T umani. Nell’insieme i nostri dati evidenziano un’associazione tra SV40 ed i linfomi non-Hodgkin. Tale associazione emerge dal rilievo di una prevalenza statisticamente significativa di anticorpi sierici contro l’agT e le VP di SV40 nei pazienti affetti da linfomi non-Hodgkin e dalla capacità trasformante di SV40 nei confronti dei linfociti T e B umani
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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