b Epstein-Barr virus (EBV) establishes a persistent latent infection in B lymphocytes and is associated with the development of numerous human tumors. Epstein-Barr nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization, has potent cell cycle deregulation capabilities, and functions as a regulator of both viral-and cellular-gene expression. We performed transcription profiling on EBNA 3C-expressing B cells and identified several chemokines and members of integrin receptor-signaling pathways, including CCL3, CCL4, CXCL10, CXCL11, ITGA4, ITGB1, ADAM28, and ADAMDEC1, as cellular target genes that could be repressed by the action of EBNA 3C alone. Chemotaxis assays demonstrated that downregulation of CXCL10 and -11 by EBNA 3C is sufficient to reduce the migration of cells expressing the CXCL10 and -11 receptor CXCR3. Gene repression by EBNA 3C was accompanied by decreased histone H3 lysine 9/14 acetylation and increased histone H3 lysine 27 trimethylation. In an EBV-positive cell line expressing all latent genes, we identified binding sites for EBNA 3C at ITGB1 and ITGA4 and in a distal regulatory region between ADAMDEC1 and ADAM28, providing the first demonstration of EBNA 3C association with cellular-gene control regions. Our data implicate indirect mechanisms in CXCL10 and CXCL11 repression by EBNA 3C. In summary, we have unveiled key cellular pathways repressed by EBNA 3C that are likely to contribute to the ability of EBV-immortalized cells to modulate immune responses, adhesion, and B-lymphocyte migration to facilitate persistence in the host.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.

BackgroundCarbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both.MethodsPrintex®90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea.ResultsAll particles had similar primary particle size, shape, hydrodynamic diameter and ζ-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 μg/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. ConclusionsOur results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-017-0189-1) contains supplementary material, which is available to authorized users.

These data indicate that distal airways might be less sensitive to IL-13-induced GC metaplasia and mucus production through lower expression of IL-13Rα1 and attenuated activation of downstream signalling. This might represent a protective strategy to prevent mucus plugging of distal airways and thus impaired ventilation of attached alveoli.

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CBNP are found in industrially produced carbon black which is preferably used for reinforcement of elastomers (e.g. in tyres) but are also present in paints, toner and batteries. CBNP may have lung cytotoxic and pro-inflammatory effects. The commercially available CBNP Printex 90 (P90) as well as the quartz DQ12 were shown to possess cytotoxic and pro-inflammatory properties in mice and human epithelial cell lines. We sought to investigate whether these particles exert similar effects in distal versus proximal airway explants of mice. Further, we investigated whether surface modifications with cancerogenic PAHs on CBNP lead to increase or decrease in the biological effects of CBNP. The impact of CBNP on different airway regions was assessed by microdissection of proximal and distal airways from mouse lungs and ex vivo stimulation for 0.5 - 48h. Our study could not show any cytotoxic effects (LDH release) on microdissected airways after incubation of up to 48h with DQ12, acetylene soot which displays various PAHs on its surface, Printex 90, as well as Printex 90 displaying the PAH 9-Nitroanthracene (NA-CBNP) or the PAH Benzo(a)pyrene (BaP-CBNP) on its surface. RT-QPCR results demonstrated that levels of IL-1-beta, KC, IFN-gamma, TNF-alpha, TGF-beta remained unchanged in mice and epithelial cell lines after 4 and 24h of particle incubation. However, IL-6 levels increased with particle concentration of any particle tested. Interestingly, this could only be observed in proximal airways. However, high variations between animals were observed. IL-6 protein did not exhibit any change after 24h of incubation. 2',7'-dichlorfluorescein-diacetate (DCFH) was used to examine oxidative stress levels. All particles showed an effect in proximal and distal airways at the highest concentration used. RT-QPCR results confirmed the induction of Glutathione peroxidase 3 (GPx-3) expression for detoxification of hydrogen peroxide and partly Glutathione reductase (GR). We can conclude that all particles tested did not result in an acute cytotoxic effect and did only slightly induce gene expression of the proinflammatory cytokine IL-6 in proximal airways. Oxidative stress was observed at the highest concentration used (50 µg/ml) whereas anti-oxidative gene expression was already induced from the lowest concentration used (0.1 µg/ml). Supported by the BMBF: joint project Carbon Black (03X0093A