1,720,955 research outputs found
Role of MMTV-like virus in human breast cancer: determination of viral presence in a primary cell line.
No full textMurine mammary tumor virus (MMTV) is an oncogenic beta-retrovirus discovered by Bittner in 1936, who demonstrated the involvement of the virus in the development of murine mammary cancer. It was shown to cause mammary carcinoma in mice following milk transmission from mother to suckling offspring. Oncogenesis occurs because the provirus integrates into the murine genome in proximity of regulatory gene promoters, modifying their expression.
Based on epidemiological observations, such a virus has also been hypothesized to be involved in the ethiology of certain types of human breast cancer. Indeed, MMTV viral sequences have been identified in DNA isolated from human mammary tumor cells, which also expressed the MMTV Env protein. Furthermore, sequences homologous to MMTV env have been observed in 38% of American patients with breast cancer. A sequence of 660 base pairs (bp) similar to MMTV env (env-like) was detected in tissue from such patients but not in normal breast tissue. These viral sequences showed a 95-98% homology to MMTV and only 56% homology with human endogenous retrovirus HERV-K10. This led to the hypothesis that there is a human virus similar to MMTV, the human mammary tumor virus (HMTV).
To assess HMTV involvement in the genesis of human breast cancer, this study was based on the analysis of a primary cell line taken from a patient with breast tumor, which was positive to HMTV genome by PCR. The aim of our research was to demonstrate the existence of such a virus, as whole virions, viral proteins or as a viral genome sequence, in this primary cell line. We attempted to confirm HMTV presence by searching for viral genome and its integration into the cell genome. In parallel, we tried to detect viral proteins such as Env and Pol, by different approaches.
Since the primary line in our hands appeared to progressively lose PCR positivity to HMTV genome, first of all, limiting dilution cloning was performed, in order to achieve positive and negative clones of the line itself. Positive and negative clones and the primary cell line were subjected to further analysis.
To assess whether viral proteins, such as Env were expressed in the cell line, Indirect Immunfluorescence analysis (IFA) was performed on clones and line with monoclonal antibodies against Env peptides. Viral expression was also determined in both clones and primary line by means of Western blot experiments, where the signal peptide of the Env protein, a protein of 14 kDa, was searched in cell lysates by means of monoclonal antibodies. While the presence of viral Env protein was found by IFA technique, Western Blot on p14 failed to detect the presence of Env.
In order to search for another viral protein in cell-free supernatants of cell line and clones, a semi-quantitative real-time PCR technique, Syber green-based PCR-enhanced reverse transcriptase (SG-PERT) assay was used to quantify reverse transcriptase (RT). During a one-step reaction, RT derived from retroviral particles will convert the MS2 RNA into cDNA and cDNA is subsequently quantified by qPCR amplification of the MS2 cDNA. SG-PERT has given ambiguous results, as the amount of RT activity detected in cell line and clone supernatants is at limit of resolution of the method. Low amount of RT expression would not be surprising since many oncoviruses are known not to produce large amounts of virions in the tissues where they induce cancer.
To demonstrate the presence of viral DNA in the genome of cell line and clones, PCR analysis was carried out. Indeed, a 220 bp fragment of HMTV env gene by PCR could be amplified from positive clones and line. To ascertain that viral DNA was integrated in cell genome and to identify the insertion point, Southern Blot, using as a probe a 600 bp sequence of MMTV env gene, was performed. It was not possible to identify the viral genome by Southern blot, but PCR amplification confirmed its presence. Again, such contradictory results are probably due to sensitivity differences in these two techniques.
Given the unclear results obtained, the next step was to search for the Gag protein. We therefore cloned two MMTV proteins that could be used to producing monoclonal antibodies to allow detection of viral Env and Gag proteins in the breast tumor line. Expression cloning of gp52 and p27 proteins of MMTV was carried out. For this purpose, the sequences coding for the two proteins were obtained from DNA of a mouse infected from MMTV and cloned in an expression vector that allowed the addition of a tail of 6 histidine residues. The 6-His tag could thus be detected by anti-histidine antibodies in Western blot and subsequently used to purify the recombinant proteins by affinity chromatography. These proteins were used to inoculate mice so as to obtain the aforesaid antibodies.
In an effort to determine the local prevalence of antibodies against MMTV, the recombinant proteins were also used to test patient sera for anti-HMTV antibodies in Western Blot. We analized 100 sera of normal individuals and 9 sera of women affected by breast cancer. Only 1% of the normal sera were positive to Gag p27 and Env gp52, whereas 33,3% (3/9) of sera from women affected by breast cancer positive to gp52 Env and 10% (1/9) positive to Gag p27 protein. These preliminary data suggest that this approach may bring very interesting results, therefore sera of patient with breast cancer will be collected and tested to obtain a statistically relevant number of cases. This approach may be used to test population for the presence of antibodies in sera for diagnostic purposes in the future
Human Wharton's jelly-derived mesenchymal stromal cells engineered to secrete Epstein-Barr virus interleukin-10 show enhanced immunosuppressive properties
No full textMesenchymal stromal cells (MSCs) modulate the immune response and represent a potential treatment for inflammatory and autoimmune diseases. We hypothesized that this feature could be potentiated by co-administering anti-inflammatory cytokines. In this article, we asked whether engineering of Wharton Jelly-derived human MSCs (WJ-hMSCs) to express an anti-inflammatory cytokine increases cell immunomodulatory properties without altering their native features
Ingegnerizzazione di cellule staminali per la messa a punto di nuovi approcci terapeutici contro il diabete mellito di tipo 1
No full textABSTRACT
Type 1 diabetes is an autoimmune disorder characterized by the reduced production of insulin due to the depletion of β cells located in the pancreatic islets of Langerhans. This disease, typically, occurs during the childhood.
Genetic, environmental and immunological factors are recognized risk factors for this pathology. The routine treatment is the injection of exogenous insulin. Many research centers, all over the world, are studying alternative treatments based on the transplantation of the pancreas (highly invasive and expensive) or the infusion of pancreatic β cells. Unfortunately most of these cells are rejected by the autoimmune response. It is therefore necessary to develop novel strategies to preserve the transplanted β cells.
The aim of this work is to develop a lentiviral vector to engineer human mesenchymal stem cells from umbilical cord (HUC-MSC) in such a way to constitutively express viral the Epstein-Barr virus interleukin-10 (vIL10). The v-IL10 is similar to the human IL-10 but is devoid of immunostimulating activity. In fact, vIL-10 inhibits cell-mediated immunity and inflammation and promotes the humoral response. The transduced cells will be then co-transplanted in the omental pouch with β cells of pancreatic islets. We have chosen a lentiviral vector derived from feline immunodeficiency virus (FIV) because is incapable to replicate in human cells.
In addition to v-IL 10, the vector encodes for the TK (thymidine kinase) from Herpes Simplex virus, which allows an in vivo screening of the cells that have incorporated the construct. These two genes will be connected by an internal ribosome entry side that makes possible the translation of both genes from the same messenger RNA.
The vector produced here is capable to release vIL-10 in supernatants of transfected HEK 293T (human embryonic kidney cell line. We are currently developing an assay to assess the functional activity of this cytokine.
This vector will be then transduced in HUC-MSC and the engineered cells will be then tested for the ability to preserve the β cells from autoimmunity
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
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
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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