Global profiling of viral and cellular non-coding RNAs in Epstein-Barr virus-induced lymphoblastoid cell lines and released exosome cargos.

Abstract The human EBV-transformed lymphoblastoid cell line (LCL), obtained by infecting peripheral blood monocular cells with Epstein–Barr Virus, has been extensively used for human genetic, pharmacogenomic, and immunologic studies. Recently, the role of exosomes has also been indicated as crucial in the crosstalk between EBV and the host microenvironment. Because the role that the LCL and LCL exosomal cargo might play in maintaining persistent infection, and since little is known regarding the non-coding RNAs of LCL, the aim of our work was the comprehensive characterization of this class of RNA, cellular and viral miRNAs, and cellular lncRNAs, in LCL compared with PBMC derived from the same donors. In this study, we have demonstrated, for the first time, that all the viral miRNAs expressed by LCL are also packaged in the exosomes, and we found that two miRNAs, ebv-miR-BART3 and ebv-miR-BHRF1-1, are more abundant in the exosomes, suggesting a microvescicular viral microRNA transfer. In addition, lncRNA profiling revealed that LCLs were enriched in lncRNA H19 and H19 antisense, and released these through exosomes, suggesting a leading role in the regulation of the tumor microenvironment

CD40 activation in human pancreatic islets and ductal cells.

Aims/hypothesis: CD40 expression on non-haematopoietic cells is linked to inflammation. We previously reported that CD40 is expressed on isolated human and non-human primate islets and its activation results in secretion of IL-8, macrophage inflammatory protein 1-beta (MIP-1β) and monocyte chemoattractant protein-1 (MCP-1) through nuclear factor-κB and extracellularly regulated kinases 1/2 pathways. The objective of this study was to identify the pattern of gene expression, and to study viability and functionality affected by CD40-CD40 ligand (CD40L) interaction in human islets. Furthermore, we have studied the CD40-mediated cytokine/chemokine profile in pancreatic ductal cells, as they are always present in human islet transplant preparations and express CD40 constitutively. Methods: CD40-CD40L gene expression modulation was studied by microarray on islet cells depleted of ductal cells. Selected genes were validated by quantitative RT-PCR. The cytokine profile in purified ductal cells was evaluated by Luminex technology, based on the use of fluorescent-coated beads, known as microspheres, and capable of multiplex detection of proteins from a single sample. Glucose-stimulated insulin secretion and islet viability were assessed by perifusion and 7-aminoactinomycin D membrane exclusion, respectively. Results: Statistical analysis of microarrays identified 30 genes exhibiting at least a 2.5-fold increase across all replicate arrays. The majority of them were related to oxidative stress/inflammation. Prominently upregulated were chemokine C-X-C motif ligand 1 (CXCL1), CXCL2 and CXCL3 belonging to the CXC family of chemokines related to IL-8. CD40-mediated CXCL1 secretion was confirmed by ELISA. The viability or in vitro function was not affected by CD40 activation. In addition to previously reported IL-8, MIP-1β and MCP-1, CD40 stimulation in ductal cells produced IL-1β, IFN-γ, TNF-α, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Conclusions/interpretation: CD40 activation in islets and ductal cells produces cytokines/chemokines with a broad-spectrum range of biological functions

Isolation and phenotypical characterization of mesenchymal stem cells from the Wharton’s jelly of preterm human umbilical cord

Extraembryonic tissues such as umbilical cord are considered a promising source of stem cells, potentially useful in therapy. Pre-term umbilical cords may be also a useful source of mesenchymal populations, given also that pre-term birth infants may develop diseases during childhood which may be reverted by a cell therapy approach. Pre-term cords can be also made available from therapeutic abortions, providing a further cell source to obtain high numbers of WJ-MSCs. Little is known about the phenotype and differentiative potential of these cells. Preterm UC were obtained following therapeutic abortions after mothers’ informed consent and processed within 12 hours from tissue collection. Characterization of cells was performed at P2 and P5, by both flow cytometry (FC) and ICC, to detect of classical MSC markers, immunomodulatory molecules, tissue-specific markers.The isolation protocol allowed to successfully derive WJ-MSCs which showed the typical morphology, and were routinely passaged up to passage 10. Multi-color flow cytometric analyses showed that isolated cells were positive for classical MSCs markers (CD29, CD44, CD73, CD90, CD105) and. negative (or weakly positive) for typical hematopoietic and endothelial markers (CD45, CD34, CD14, CD68, CD39 and CD31). In addition, we demonstrated the expression of tissue specific markers, both endodermal (CK18, CK19, alpha-fetoprotein and albumin) and neuro-ectodermal (nestin). This may indicate the potential of preterm WJ-MSC to undergo multiple differentiation pathways, as demonstrated for cells isolated from term UC. Preterm WJ-MSCs showed MHC class I expression (but not class II), suggesting hypoimmunogenic properties for these cells. Moreover, B7H3 expression should favor immune tolerance by the host following cellular transplantation. ICC allowed confirming part of the FC data and was used to assess the expression of further antigens. Present data demonstrate that preterm WJ-MSCs can be isolated and expanded, with high reproducibility. these cells do express classical MSC markers and immunomodulatory molecules, independently from the underlying pathology which led to abortion