Objective. Although monosodium urate monohydrate (MSU) crystals have been recognized since the 18th century as the etiologic agent of gout, it is still unknown why certain hyperuricemic individuals remain asymptomatic, and how an acute attack of gout spontaneously resolves. We hypothesized that mononuclear phagocytes hold the key to these questions, and that the state of monocyte/macrophage differentiation is critical.Methods. Human peripheral blood monocytes were differentiated for 1-7 days in vitro and examined with respect to 1) uptake of MSU crystals, 2) expression of macrophage, dendritic cell, and activation markers, 3) secretion of tumor necrosis factor ␣ (TNF␣), interleukin 1␤ (IL-1␤), IL-6, and IL-10, 4) activation of endothelial E-selectin expression, and 5) enhancement of secondary neutrophil recruitment by endothelial cells.Results. MSU crystals induced TNF␣, IL-1␤, and IL-6 (but not IL-10) secretion in undifferentiated monocytes, which in turn promoted endothelial cell E-selectin expression and secondary neutrophil capture under shear flow. In contrast, differentiation over 3-5 days led to development of a noninflammatory phenotype characterized by a lack of proinflammatory cytokine secretion, lack of endothelial cell activation, and lack of secondary neutrophil recruitment. Acquisition of the noninflammatory phenotype correlated with expression of macrophage antigen but not with expression of dendritic cell marker or activation marker. Monocytes and macrophages were similarly phagocytic, and a control particle, zymosan, elicited secretion of the full panel of cytokines in both cell types. However, coincubation with MSU led to a significant suppression of zymosaninduced TNF␣ secretion (P ‫؍‬ 0.009) from macrophages but not monocytes.Conclusion. These findings imply that differentiated macrophages provide a safe-disposal mechanism for the removal of inflammatory urate crystals. This may be of clinical relevance to the maintenance of asymptomatic hyperuricemia and the resolution of acute gout.

Objective. It has previously been shown that as monocytes differentiate into macrophages, they lose the ability to secrete proinflammatory cytokines in response to monosodium urate monohydrate (MSU) crystals. The purpose of this study was to investigate whether MSU crystals induce macrophages to secrete antiinflammatory factor instead.Methods. Human monocyte or macrophage isolates were prepared from samples obtained from healthy volunteer donors either by differentiation of blood monocytes in vitro or by collecting cells from skin blisters during the early or late phase of the dermal inflammatory response to cantharidin. Monocyte or macrophage isolates were then incubated with MSU crystals for 24 hours, and culture supernatants were assayed for candidate antiinflammatory mediators (by enzyme-linked immunosorbent assay) and for the capacity to activate or suppress endothelial cell E-selectin expression and secondary neutrophil recruitment under shear flow.Results. Analysis of supernatants from in vitrodifferentiated macrophages revealed that transforming growth factor ␤1 ( The mechanisms behind the spontaneous resolution of gout remain incompletely explained. In the absence of clinical intervention, acute gouty arthritis usually resolves within a few days. However, monosodium urate monohydrate (MSU) crystals can persist in joints following acute inflammation and have been identified within mononuclear cells in synovial fluid aspirated from clinically quiescent knee and metatarsophalangeal joints (1-3). A possible beneficial role of macrophages in gout has been demonstrated with the observation that mouse macrophage cell lines and human in vitro-differentiated macrophages ingest MSU crystals without proinflammatory cytokine secretion or endothelial cell activation (4,5). Such quiescent behavior is in marked contrast to less well-differentiated monocytic isolates, which secrete tumor necrosis factor ␣ (TNF␣), interleukin-1␤ (IL-1␤), IL-6, and IL-8, activate E-selectin expression, and promote secondary neutrophil capture by endothelial cells under physiologic flow in response to MSU crystal uptake (5-9).

The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.

Objective. We have hypothesized that the process of monocyte to macrophage differentiation may alter the inflammatory response of mononuclear phagocytes to the uptake of monosodium urate monohydrate (MSU) crystals.Methods. Eight mouse monocyte/macrophage cell lines were arranged in increasing order of differentiation, as judged by expression of the macrophage markers F4/80 and BM 8 and by phagocytic capacity. Secretion of tumor necrosis factor ␣ (TNF␣) in response to MSU was measured by enzyme-linked immunosorbent assay.Results. The panel of monocyte/macrophage cell lines revealed a close linkage between the state of differentiation and the capacity of the cells to ingest MSU crystals. TNF␣ production, however, was not linked to phagocytic ability. Peak TNF␣ levels were synthesized by cells at an intermediate state of differentiation (3.2-14.1 ng/ml), whereas mature macrophages, which efficiently phagocytosed crystals, did not secrete TNF␣. Mature cell lines produced TNF␣ when stimulated with zymosan (5.9-6.2 ng/ml), but this was abolished by coincubation with MSU crystals. Suppression of the zymosan response was not due to apoptosis or steric hindrance by MSU crystals. Culture supernatants from mature macrophages did not stimulate endothelial cell activation, in contrast to MSU-treated cells at an earlier stage of differentiation, which stimulated intercellular adhesion molecule 1 expression on sEND endothelioma cells through the release of TNF␣ (inhibited 80.6% by anti-TNF␣).Conclusion. We demonstrated that phagocytosis and TNF␣ production are distinct events in the response of mononuclear phagocytes to urate crystals, and these events can be distinguished at the level of macrophage differentiation. The noninflammatory removal of urate crystals by mature macrophages defines a new pathway that may be important in controlling the development of acute gout in patients with hyperuricemia.Acute gouty arthritis is a self-limiting inflammatory response to the intraarticular deposition of monosodium urate monohydrate (MSU) microcrystals. Previous studies investigating the effect of MSU crystals in mononuclear phagocytes have focused primarily on the ability of peripheral blood monocytes or monocytic cell lines to secrete proinflammatory cytokines such as tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), . Investigators in our group have further shown that TNF␣ and IL-1 released by peripheral blood monocytes can activate vascular endothelial cell expression of E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1, thereby stimulating both the recruitment of leukocytes to the site of crystal deposition and the amplification of the inflammatory response (6-8).Hyperuricemia and precipitation of inflammatory microcrystals in synovial fluid per se are not always predictive of acute synovitis, as the severity of disease Supported by grants from the British Heart Foundation and Glaxo Wellcome PLC.

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.