Background-During systemic inflammation, activation of vascular endothelium by proinflammatory cytokines leads to hypotension, microvascular thrombosis, and organ damage. Recent data suggest a link between coagulation and inflammation through the activated protein C (APC) pathway. We studied gene expression profiles in human coronary artery endothelial cells (HCAECs) exposed to proinflammatory stimuli and the influence of APC on expression of candidate genes regulated by these stimuli. Methods and Results-HCAECs were stimulated with interleukin-1, interferon-␥, and tumor necrosis factor-␣. In gene expression profiling, 400 of 8400 genes were regulated Ͼ2-fold. Verification of selected candidate genes was achieved by measuring expression of mRNA species by real-time polymerase chain reaction, cytokine secretion by ELISA, and metabolites of tetrahydrobiopterin (BH4) biosynthesis by high-performance liquid chromatography. BH4 synthesis, interleukin-6, interleukin-8, monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were downregulated by APC at the transcriptional and protein level. Endothelial nitric oxide synthase, endothelial adhesion molecule, and vascular cell adhesion molecule-1 were not affected by APC. Activities of transcription factors c-Fos, FosB, and c-Rel were inhibited by APC in inflamed HCAECs. Conclusions-Our study revealed a novel antiinflammatory mechanism of APC-dependent gene regulation in HCAECs since c-Fos-dependent induction of MCP-1 and ICAM-1 was suppressed. APC downregulates expression and activity of genes related to inflammation, most pronounced under intermediate or mild inflammatory conditions. (Circulation.
SUMMARYBALB/c mice were infested with Ixodes ricinus larvae, nymphs or adults. Expression of IL-4 and IFN-g mRNA in axillary and brachial draining lymph node cells were measured by competitive quantitative reverse transcription-polymerase chain reaction 9 days after the beginning of primary-infestation. IL-4 mRNA was always higher than that of IFN-g mRNA for all tick instars. Moreover, IL-4 mRNA expression progressively increased during nymphal primary-infestation with a high burst of expression 7 days after the beginning of infestation. No evolution of IFN-g mRNA expression was detected. Draining lymph node cells of infested BALB/c produced higher level of IL-4 than IFN-g following in vitro restimulation with adult tick saliva, salivary gland extract (SGE) or with five selected different chromatographic fractions of SGE. Anti-tick IgG1 antibodies but no IgG2a were detected in BALB/c pluriinfested with IX ricinus nymphs, which confirmed the Th2 polarization of the immune response.
Background-Previous studies have provided evidence for the importance of platelet-derived nitric oxide (NO) for the regulation of hemostasis. Tetrahydrobiopterin (BH 4 ) is an essential cofactor and regulator of NO synthase activity in the vasculature; however, it is as yet unknown whether platelets dispose over a functional BH 4 synthesis. Methods and Results-We quantified mRNA expression of genes involved in BH 4 synthesis, measured enzymatic activities, and determined intraplatelet levels of pteridines in platelets from healthy volunteers and from patients treated for prolonged periods of time with glucocorticoids. Freshly isolated platelets from healthy volunteers show functional BH 4 synthesis, as evidenced by the presence of mRNA species and enzymatic activity of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase. Biopterin was the major intraplatelet pteridine, whereas no neopterin was found. mRNA expression and enzymatic activity of GTPCH were undetectably low in platelets that had been stored for 5 days, and no pteridines were found in these platelets. Freshly isolated platelets from patients treated with glucocorticoids had decreased mRNA expression and activity of GTPCH compared with platelets from healthy volunteers. Conclusions-Human platelets dispose over a functional de novo BH 4 synthesis. Furthermore, our results indicate the potential of external factors, eg, prolonged storage or glucocorticoid therapy, to significantly affect BH 4 synthesis within platelets. Together, these findings offer new insights into the biology and pathobiology of platelet function in humans.
Our data show that PTPS induction is necessary for optimized BH4 synthesis in cytokine-stimulated HCAECs and point to IL-1beta as a leading cytokine in this process.
Summary Background There is limited information on the effects of statins on the outcomes of liver transplantation (LT), regarding either their use by LT recipients or donors. Aim To analyse the association between statin exposure and recipient and graft survival. Methods We included adult LT recipients with deceased donors in a nationwide prospective database study. Using a multistate modelling approach, we examined the effect of statins on the transition hazard between LT, biliary and vascular complications and death, allowing for recurring events. The observation time was 3 years. Results We included 998 (696 male, 70%, mean age 54.46 ± 11.14 years) LT recipients. 14% of donors and 19% of recipients were exposed to statins during the study period. During follow‐up, 141 patients died; there were 40 re‐LT and 363 complications, with 66 patients having two or more complications. Treatment with statins in the recipient was modelled as a concurrent covariate and associated with lower mortality after LT (HR = 0.35; 95% CI 0.12–0.98; p = 0.047), as well as a significant reduction of re‐LT (p = 0.004). However, it was not associated with lower incidence of complications (HR = 1.25; 95% CI = 0.85–1.83; p = 0.266). Moreover, in patients developing complications, statin use was significantly associated with decreased mortality (HR = 0.10; 95% CI = 0.01–0.81; p = 0.030), and reduced recurrence of complications (HR = 0.43; 95% CI = 0.20–0.93; p = 0.032). Conclusions Statin use by LT recipients may confer a survival advantage. Statin administration should be encouraged in LT recipients when clinically indicated.
Implementation of regenerative medicine in the clinical setting requires not only biological inventions, but also the development of reproducible and safe method for cell isolation and expansion. As the currently used manual techniques do not fulfill these requirements, there is a clear need to develop an adequate robotic platform for automated, large-scale production of cells or cell-based products. Here, we demonstrate an automated liquid-handling cell-culture platform that can be used to isolate, expand, and characterize human primary cells (e.g., from intervertebral disc tissue) with results that are comparable to the manual procedure. Specifically, no differences could be observed for cell yield, viability, aggregation rate, growth rate, and phenotype. Importantly, all steps-from the enzymatic isolation of cells through the biopsy to the final quality control-can be performed completely by the automated system because of novel tools that were incorporated into the platform. This automated cell-culture platform can therefore replace entirely manual processes in areas that require high throughput while maintaining stability and safety, such as clinical or industrial settings.
Tissue engineering as an emerging biotechnology sector aims at the in vitro regeneration of diseased tissues and promises to profoundly change medical practice, offering the possibility of regenerating tissues and organs instead of just repairing them (regenerative medicine). Improved healing processes and a higher quality of life are the expected results. This article gives an overview of different technologies for regenerative medicine and presents results of our own current applied research and development. A recent project was successfully closed with the development of a natural biomaterial for soft tissue oral defects. The establishment of an in vitro bioreactor system enabled us to simulate the mechanical and biological environment in a healing wound and to investigate the suitability of different implant materials for the oral tissue regeneration. Moreover, focusing the attention on an alternative method for the intervertebral disc (IVD) regeneration, we established a new tissue engineered approach, based on the three-dimensional (3D) culture of autologous human IVD cells into a polyurethane (PU)-fibrin composite. IVD cells were able to proliferate and, thanks to the 3D conditions, to differentiate expressing the typical native tissue markers. The development of an automated platform was the goal of an additional project, to standardize the cell culture technology, increase the bio-safety and reduce the production costs, moving tissue engineering nearer to clinical application.
Background: Deceased-donor kidney allografts are exposed to injury during ex vivo transport due to the lack of blood oxygen supply. Hypothermic machine perfusion (HMP) is effective in reducing the risk of delayed graft function in kidney transplant recipients compared to standard cold storage. However, there is no software implementation available to read, process and analyse HMP data for state-of-the-art visualization and quality control in an analytics dashboard. Results: We implemented the tool EXAM (ex vivo allograft monitoring) which includes an interactive analytics dashboard. The backbone is a collection of functions in the R programming language to read, process and analyse HMP data from the LifePort kidney transporter (Organ Recovery Systems, USA). Time series for pressure, flow rate, organ resistance and temperature are visualized and relevant statistical indicators have been developed. We highlight a few showcases for data analysis and quality control. Conclusions: Allograft monitoring during transport and retrospective HMP data analysis are essential for quality control and novel research. EXAM is freely available software written in the R programming language that can be easily deployed by statisticians and data scientists to enable quality control at transplant organizations that use LifePort kidney transporters. The open development enables collaboration to further extend EXAM, e.g. integrating data from other kidney transporters or even form other organs into a unified state-of-the-art monitoring tool.
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