Uptake and degradation of mast-cell granules by mouse peritoneal macrophages

35S-labelled mast-cell granules isolated from mouse mastocytomas were added to mouse macrophages in vitro. The granules were avidly phagocytosed, and subsequently the radioactivity was released to the medium as inorganic [35S]sulphate. After pulse-labelling, a total of about 80% of the cell-associated radioactivity was thus released in the course of 24 h, indicating an extensive breakdown of the sulphated polysaccharides, mainly heparin, present in the granules. The uptake of the mast-cell granules caused pronounced, but reversible, spreading of the macrophages

Evaluation of pre-PCR processing approaches for enumeration of Salmonella enterica in naturally contaminated animal feed

Three pre-PCR processing strategies for the detection and/or quantification of Salmonella in naturally contaminated soy bean meal were evaluated

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

Secretion of IL-1β from monocytes in gout is redox independent

The proinflammatory cytokine interleukin-1β (IL-1β) plays important roles in immunity but is also implicated in autoimmune disease. The most well-established mechanism of IL-1β secretion is via activation of the NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome which requires an initial priming signal followed by an activating signal. However, the precise mechanism by which the inflammasome is activated remains unclear. The role of reactive oxygen species (ROS) in this process is contradictory, with some studies suggesting that ROS are crucial while others describe opposite effects. In this study, we evaluated the effects of oxidative stress on IL-1β secretion. Gout is a disease driven solely by IL-1β secretion in response to monosodium urate (MSU) crystals which form during periods of hyperuricemia and thus presents an opportunity to study factors contributing to IL-1β secretion. Sera and monocytes were isolated from patients with gout to determine whether differences in antioxidant status could explain the susceptibility of these individuals to gout attacks. In addition, sera and monocytes were collected from patients with chronic kidney disease (CKD) for comparison as this condition is associated with high levels of oxidative stress and disturbances in serum uric acid levels. There were differences in some aspects of antioxidant defenses in gout patients and these were mainly due to higher serum uric acid. Monocytes from gout patients were more responsive to priming, but not activation, of the NLRP3 inflammasome. However, expression of the components of the NLRP3 inflammasome were unaffected by priming or activation of the inflammasome, nor were these expression levels differentially regulated in gout patients. Inhibition of ROS by N-Acetyl Cysteine inhibited TLR2-induced priming of the NLRP3 inflammasome, but had no effect on MSU-induced activation. Together these findings demonstrate that oxidative stress only affects priming of the NLRP3 inflammasome but does not influence activation