Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling

Lipopolysaccharide (LPS) from the cell envelope of Gram-negative bacteria is a principal cause of the symptoms of sepsis. LPS has been reported to modulate the function of platelets although the underlying mechanisms of LPS action in these cells remain unclear. Platelets express the Toll-like receptor 4 (TLR4) which serves as a receptor for LPS, although the potential role of TLR4 and associated cell signalling in controlling platelet responses to LPS has not been extensively explored. In this study, we therefore investigated the actions of LPS prepared from different strains of Escherichia coli on platelet function, the underlying signalling mechanisms, and the potential role of TLR4 in orchestrating these. We report that LPS increased the aggregation of washed platelets stimulated by thromboxane (U46619) or GPVI collagen receptor agonists, effects that were prevented by a TLR4 antagonist. Associated with this, LPS enhanced fibrinogen binding, P-selectin exposure and reactive oxygen species (ROS) release. Increase of ROS was found to be important for the actions of LPS on platelets, since these were inhibited in the presence of superoxide dismutase or catalase. The effects of LPS were associated with phosphorylation of Akt, ERK1/2 and PLA2 in stimulated platelets, and inhibitors of PI3-kinase, Akt and ERK1/2 reduced significantly LPS enhanced platelet function and associated ROS production. Furthermore, inhibition of platelet cyclooxygenase or the thromboxane receptor, revealed an important role for thromboxane A2. We therefore conclude that LPS increases human platelet activation through a TLR4-PI3K-Akt-ERK1/2-PLA2 -dependent pathway that is dependent on ROS and TXA2 formation

Polycomb Repressive Complex 2 Regulates Genes Necessary for Intestinal Microfold Cell (M Cell) Development

BACKGROUND & AIMS: Microfold cells (M cells) are immunosurveillance epithelial cells located in the Peyer's patches (PPs) in the intestine and are responsible for monitoring and transcytosis of antigens, microorganisms, and pathogens. Mature M cells use the receptor glycoprotein 2 (GP2) to aid in transcytosis. Recent studies have shown transcription factors, Spi-B and SRY-Box Transcription Factor 8 (Sox8). are necessary for M-cell differentiation, but not sufficient. An exhaustive set of factors sufficient for differentiation and development of a mature GP2+ M cell remains elusive. Our aim was to understand the role of polycomb repressive complex 2 (PRC2) as an epigenetic regulator of M-cell development. Estrogen-related-receptor gamma (Esrrg), identified as a PRC2-regulated gene, was studied in depth, in addition to its relationship with Spi-B and Sox8. METHODS: Comparative chromatin immunoprecipitation and global run-on sequencing analysis of mouse intestinal organoids were performed in stem condition, enterocyte conditions, and receptor activator of nuclear factor kappa B ligand-induced M-cell condition. Esrrg, which was identified as one of the PRC2-regulated transcription factors, was studied in wild-type mice and knocked out in intestinal organoids using guide RNA's. Sox8 null mice were used to study Esrrg and its relation to Sox8. RESULTS: chromatin immunoprecipitation and global run-on sequencing analysis showed 12 novel PRC2 regulated transcription factors, PRC2-regulated Esrrg is a novel M-cell-specific transcription factor acting on a receptor activator of nuclear factor kappa B ligand-receptor activator of nuclear factor kappa B-induced nuclear factor-kappa B pathway, upstream of Sox8, and necessary but not sufficient for a mature M-cell marker of Gp2 expression. CONCLUSIONS: PRC2 regulates a significant set of genes in M cells including Esrrg, which is critical for M-cell development and differentiation. Loss of Esrrg led to an immature M-cell phenotype lacking in Sox8 and Gp2 expression. Transcript profiling: the data have been deposited in the NCBI Gene Expression Omnibus database (GSE157629).Peer reviewe

Optimization of a genotyping screening based on hydrolysis probes to detect the main mutations related to Leber hereditary optic neuropathy (LHON)

CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOLeber hereditary optic neuropathy (LHON) is a mitochondrial inherited disease characterized by bilateral vision problems, such as reduced visual acuity, dyschromatopsia, and central or centrocecal scotoma. Of these cases, 95% are caused by three mutations in mitochondrial DNA (mtDNA): m.G11778A, followed by m.T14484C and m. G3460A. The remaining 5% of cases of LHON are caused by rare mutations also present in mtDNA. Although conventional molecular tools for molecular screening of LHON are becoming popular, in most cases these tools are still expensive and time-consuming and are difficult to reproduce. Therefore, to meet the need for more accurate, faster, and cheaper techniques for molecular screening, as well as make it more accessible, we used the high-throughput method TaqMan (R) OpenArray (TM) Genotyping platform for developing a customized high-throughput assay for the three main mutations related to LHON. Methods: The assay was performed for 87 individuals diagnosed with LHON or acquired optic neuropathy of unknown origin. The three main mutations were screened using the customized assay with the TaqMan (R) OpenArray (TM) Genotyping platform, and all reactions were performed in triplicate. The positive and negative results were revalidated with restriction fragment length polymorphism PCR (RFLP-PCR) and Sanger sequencing. Results: The main mutations related to LHON were detected in 34 patients with genotyping reactions, of which 27 cases had the m. G11778A mutation, and seven had the m. T14484C mutation. Conclusions: The TaqMan (R) OpenArray (TM) Genotyping platform was shown to be an effective tool for molecular screening of the most common mutations related to LHON without presenting false positive or negative results for the analyzed mutations. In addition, this tool can be considered a cheaper, faster, and more accurate alternative for molecular screening of LHON mutations than PCR and Sanger sequencing, as 94 genotyping reactions can be performed within 6 h and specific TaqMan probes are used23495503CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informação2014/10188-