Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD) and emphysema. Although cigarette smoke represses cellular proliferation, the molecular mechanisms underlying this phenomenon are unknown. CCAAT/enhancer-binding proteins (C/EBPs) are key regulators of cell cycle progression, differentiation and pro-inflammatory gene expression, are regulated predominantly at the translational level and may be involved in the pathogenesis of COPD. The aim of this study was to assess the effect of cigarette smoke on proliferation and the expression and translational regulation of C/EBPa and C/EBPb in nondiseased primary human lung fibroblasts.Fibroblasts were exposed to cigarette smoke-conditioned medium (10% and 20% for 24 h). Proliferation was determined by [ 3 H]thymidine incorporation. Protein expression levels were determined by immunoblotting and translation was monitored using a translation control reporter system.Cigarette smoke significantly reduced fibroblast proliferation and significantly upregulated fulllength C/EBPa and C/EBPb proteins due to a shift in the translational control of CEBPA and CEBPB mRNAs. This shift involved the re-initiation of mRNA translation via the regulatory upstream open reading frame, which coincided with increased interleukin-8 release and a decrease in functional elastin level.These findings provide a novel mechanism to understanding the tissue remodelling observed in the lungs of COPD patients.
Introduction: This article aims to identify best practices, improve risk controls, and aid regulatory agencies in developing guidance for environmental and biosafety risk assessment for commercial-scale cell and gene therapy manufacturing. Methods: A cross-functional team should start with hazard classification and testing requirements for materials used or generated by the process and process hazard characterization. Results: The team develops a safety profile of the process to mitigate risks, including: product biological contamination risk and process controls, including raw materials, facilities, operator and environmental controls, and method of detection; a technical review of the process to evaluate the operational and engineering controls; monitoring systems to mitigate the risk of failure and/or breach of the system, preventing the release of material to the facility or operator exposure; site sanitization strategy and facility containment measures, including engineering designs, air handling systems, spill containment measures, surface cleanability, waste flows, and decontamination practices; a review of site practices, including process, employee, material and waste flows, staff training, controlled access, operator gowning, and emergency response plans/measures. Discussion: The cross-functional team should regularly reconvene to provide solutions for enhanced process control, process life-cycle management, monitor assumptions, and track performance. The plan must be revised following any relevant failure event or process change. Conclusion: A risk assessment template is shared to bring to the reader’s attention the complexity of commercial-scale manufacturing, areas to assess, potential questions to ask, and other pertinent parties who may input to the risk assessment.
Circulating B lymphocytes are the major reservoir of KSHV infection in infected subjects. However, B cell lines and primary B cells are resistant to direct KSHV infection in vitro. In addition, primary B cells are difficult to propagate for more than a few days. In this study, we combined a novel primary B cell propagation method with efficient infection mediated by dendritic cells to study KSHV de novo infection of primary B cells in vitro.Primary monocyte-derived dendritic cells (MDDCs) or plasmacytoid dendritic cells (pDCs) were pulsed with KSHV for 4 hours at which point KSHV DNA was readily detectable. Uptake of KSHV was significantly reduced by pre-incubating cells with antibodies to integrins α3, β1 and DC-SIGN. Autologous B cells were grown on a feeder layer of irradiated NIH3T3 cells transduced with a human CD40L retroviral vector. KSHV+ DCs were co-cultivated with primary B cells for 4-8 hours and then separated by CD19+ immunomagnetic isolation. B cell cultures were maintained on feeder cells for >30 days and monitored for KSHV infection.Efficient KSHV infection of primary B cells was mediated by both MDDCs and pDCs. KSHV LANA protein (ORF73) was detected by IFA in 2-15 percent of B cells through day 14. Viral gene expression analysis using a KSHV whole genome virus array showed establishment of latent KSHV infection followed by spontaneous reactivation of lytic viral replication in the primary B cell cultures.These studies suggest that dendritic cells play an important role in the transmission and pathogenesis of KSHV in infected subjects as well as demonstrating a powerful in vitro model for studying KSHV infection of B cells.
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