Precipitation of soluble uric acid is necessary for in vitro activation of the NLRP3 inflammasome in primary human monocytes

Objective. To investigate the effects of soluble uric acid (UA) on expression and activation of the NOD-like receptor (NLR) pyrin domain containing protein 3 (NLRP3) inflammasome in human monocytes to elucidate the role of hyperuricemia in the pathogenesis of gout. Methods. Primary human monocytes and the THP-1 human monocyte cell line were used to determine the effects of short- and long-term exposure to UA on activation of the NLRP3 inflammasome and subsequent interleukin-1β (IL-1β) secretion by enzyme linked immunosorbent assay (ELISA) and cell-based assays. Expression of key NLRP3 components in monocytes from patients with a history of gout were analysed by quantitative PCR. Results. Precipitation of UA was required for the activation of the NLRP3 inflammasome and subsequent release of IL-1β in human monocytes. Neither monosodium urate (MSU) crystals nor soluble UA had any effect on activation of the transcription factor, NF-κB. Prolonged exposure of monocytes to soluble UA did not alter these responses. However, both MSU crystals and soluble UA did result in a 2-fold increase in reactive oxygen species (ROS). Gout patients (n=15) had significantly elevated serum UA concentrations compared to healthy individuals (n=16), yet secretion of IL-1β and expression of NLRP3 inflammasome components in monocytes isolated from these patients were not different from healthy controls. Conclusion. Despite recent reports indicating that soluble UA can prime and activate the NLRP3 inflammasome in human peripheral blood mononuclear cells (PBMCs), precipitation of soluble UA into MSU crystals is essential for in vitro NLRP3 signalling in primary human monocytes

Rapid and Efficient Clearance of Blood-borne Virus by Liver Sinusoidal Endothelium

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity

Isolation of chromosome clusters from metaphase-arrested HeLa cells

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosomes from a cell remain together in a bundle which we call a “metaphase chromosome cluster”. Cells are arrested to 90–95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a Percoll TM density gradient. — This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca ++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47359/1/412_2004_Article_BF00327351.pd

Preparation of sealed Torpedo marmorata membrane fragments suitable for quantitative tracer flux studies

Bernhardt J, Moss K, Luckinger RM, Neumann E. Preparation of sealed Torpedo marmorata membrane fragments suitable for quantitative tracer flux studies. FEBS Letters. 1981;134(2):245-248