Jacquie L. Harper

Resident macrophages initiating and driving inflammation in a monosodium urate monohydrate crystal-induced murine peritoneal model of acute gout.

OBJECTIVE To determine whether infiltrating monocytes, neutrophils, or resident macrophages contribute to the early inflammatory response to monosodium urate monohydrate (MSU) crystals in vivo. METHODS MSU crystal-induced inflammation was monitored using a peritoneal model of acute gout. The production of proinflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha [TNFalpha], IL-6) by resident macrophages, infiltrating monocytes, and neutrophils during the onset of gout was determined by flow cytometry. Infiltrating and resident peritoneal cells were cultured with MSU crystals ex vivo, and proinflammatory cytokine production was determined by multiplex cytokine array. Activated macrophages on the visceral epithelial lining of the peritoneum were identified by immunofluorescence histochemistry. The inflammatory immune response to MSU crystals was then compared with the inflammatory response in mice depleted of resident macrophages by pretreatment with clodronate liposomes. RESULTS The production of cytokines in vivo preceded the influx of Gr-1(intermediate)7/4+ monocytes. Monocytes and neutrophils recruited during the inflammatory phase of the response to MSU crystals failed to produce proinflammatory cytokines either in vivo, or ex vivo following restimulation with MSU crystals. Stimulation of the naive peritoneal resident cell population with MSU crystals ex vivo resulted in positive staining of resident macrophages for the proinflammatory cytokines IL-1beta, TNFalpha, and IL-6. Depletion of the resident macrophage population resulted in a significant decrease in both MSU crystal-induced neutrophil infiltration and proinflammatory cytokine production in vivo despite the presence of infiltrating monocytes. CONCLUSION These data indicate that resident macrophages, rather than infiltrating monocytes or neutrophils, are important for initiating and driving the early proinflammatory phase of acute gout.

A Role for Gut Microbiota and the Metabolite‐Sensing Receptor GPR43 in a Murine Model of Gout

Host–microbial interactions are central in health and disease. Monosodium urate monohydrate (MSU) crystals cause gout by activating the NLRP3 inflammasome, leading to interleukin‐1β (IL‐1β) production and neutrophil recruitment. This study was undertaken to investigate the relevance of gut microbiota, acetate, and the metabolite‐sensing receptor GPR43 in regulating inflammation in a murine model of gout.

Pharmacological concentrations of ascorbate radiosensitize glioblastoma multiforme primary cells by increasing oxidative DNA damage and inhibiting G2/M arrest

Glioblastoma multiforme (GBM) has a very poor prognosis because of its chemo- and radiation therapy resistance. Here we investigated the ability of pharmacological concentrations of ascorbate to radiosensitize primary cells isolated from six GBM patients, mouse astrocytoma cells, and mouse astrocytes. We measured cell viability by trypan blue exclusion, generation of double-stranded DNA breaks by H2AX phosphorylation using fluorescently labeled antibodies and FACS analysis, apoptosis by annexin V/propidium iodide staining, inhibition of autophagy by 3-methyladenine, and cell cycle progression by propidium iodide staining of permeabilized cells. We showed that 5 mM ascorbate in combination with 6 Gy radiation killed more GBM primary cells by generating significantly more double-stranded breaks than either treatment alone (p<0.05). Combined treatment affected viability and double-stranded break generation in normal astrocytes to a much smaller extent. Radiation, but not 5 mM ascorbate, caused G2/M arrest in GBM cells and ascorbate prevented radiation-induced G2/M arrest in combined treatment. Cell death in response to 5 mM ascorbate or combination treatment was not mediated by apoptosis or autophagy. In conclusion, pharmacological concentrations of ascorbate radiosensitize GBM primary cells to a much greater extent than astrocytes; this large therapeutic ratio may be of clinical significance in radiation-resistant cancers.

Didemnidines A and B, Indole Spermidine Alkaloids from the New Zealand Ascidian Didemnum sp.

Two new indole spermidine alkaloids, didemnidines A (1) and B (2), have been isolated from the New Zealand ascidian Didemnum sp. The structures of the metabolites, determined by analysis of 2D NMR spectra and confirmed via synthesis, embody an indole-3-glyoxylamide moiety linked to the N(1) position of spermidine, the latter motif being particularly rare among marine natural products. Didemnidine B and a synthetic precursor exhibited mild in vitro growth inhibition of Plasmodium falciparum with IC(50)s of 15 and 8.4 μM, respectively.

Long-Chain Lipids Are Required for the Innate Immune Recognition of Trehalose Diesters by Macrophages

Going to any length? Trehalose diesters of various chain lengths have been synthesised in order to determine the effect of lipid length on innate immune recognition, as determined by NO and cytokine production by macrophages. In this work, we show that longer lipids (C(20) -C(26)) are required for macrophage activation, with C(22) giving optimal activity.

Colchicine suppresses neutrophil superoxide production in a murine model of gouty arthritis: a rationale for use of low-dose colchicine.

Background and purpose: When used to treat gouty arthritis, colchicine is believed to work by inhibiting microtubule‐dependent cell infiltration. However, in vitro, colchicine also reduces monosodium urate (MSU)‐induced superoxide production by neutrophils. Our study aimed to compare the effects of colchicine on neutrophil superoxide production and infiltration in an in vivo model of acute gouty inflammation.

Indigenous New Zealand honeys exhibit multiple anti-inflammatory activities.

Recent evidence suggests a potential role for honeys in mediating clinical inflammation and tissue damage. Here, we investigated the anti-inflammatory activity of a selection of previously untested indigenous New Zealand (NZ) honeys. We found that several, but not all, New Zealand rewarewa, manuka and kanuka honey samples exhibited potent, dose-dependent reduction of human neutrophil superoxide production in vitro. This inhibitory activity did not correlate with levels of known phenolic-based free radical scavengers. Furthermore, the active honeys did not scavenge superoxide generated in a cell-free xanthine/xanthine oxidase assay. In C57BL/6 J mice, topical application of manuka and rewarewa honey samples with the highest in vitro activity suppressed arachidonic acid-induced ear oedema, and rewarewa honey suppressed both oedema and leukocyte (monocyte and neutrophil) infiltration. Together, these findings demonstrate that some indigenous NZ honeys exhibit clinically relevant anti-inflammatory activity. Further investigation is warranted to identify the active component(s) and mechanisms responsible for these activities and to determine potential applications for anti-inflammatory honeys in the topical treatment of clinical inflammation.

Activation and route of administration both determine the ability of bone marrow-derived dendritic cells to accumulate in secondary lymphoid organs and prime CD8+ T cells against tumors

AbstractAimsTo examine the effects of route of administration and activation status on the ability of dendritic cells (DC) to accumulate in secondary lymphoid organs, and induce expansion of CD8+ T cells and anti-tumor activity.MethodsDC from bone marrow (BM) cultures were labeled with fluorochromes and injected s.c. or i.v. into naïve mice to monitor their survival and accumulation in vivo. Percentages of specific CD8+ T cells in blood and delayed tumor growth were used as readouts of the immune response induced by DC immunization. ResultsThe route of DC administration was critical in determining the site of DC accumulation and time of DC persistence in vivo. DC injected s.c. accumulated in the draining lymph node, and DC injected i.v. in the spleen. DC appeared in the lymph node by 24 h after s.c. injection, their numbers peaked at 48 h and declined at 96 h. DC that had spontaneously matured in vitro were better able to migrate compared to immature DC. DC were found in the spleen at 3 h and 24 h after i.v. injection, but their numbers were low and declined by 48 h. Depending on the tumor cell line used, DC injected s.c. were as effective or more effective than DC injected i.v. at inducing anti-tumor responses. Pre-treatment with LPS increased DC accumulation in lymph nodes, but had no detectable effect on accumulation in the spleen. Pre-treatment with LPS also improved the ability of DC to induce CD8+ T cell expansion and anti-tumor responses, regardless of the route of DC administration.Conclusions Injection route and activation by LPS independently determine the ability of DC to activate tumor-specific CD8+ T cells in vivo.

Monosodium urate monohydrate crystal–recruited noninflammatory monocytes differentiate into M1-like proinflammatory macrophages in a peritoneal murine model of gout

OBJECTIVE To profile monosodium urate monohydrate (MSU) crystal-recruited monocyte inflammatory function during the course of in vivo differentiation, in a murine model of peritoneal MSU crystal-induced inflammation. METHODS C57BL/6J mice were injected intraperitoneally with MSU crystals, and the peritoneal cells were harvested at different time points. The MSU crystal-recruited monocyte/macrophage population was analyzed for the expression of differentiation and activation markers, cytokine production following MSU crystal restimulation ex vivo and in vivo, expression of NLRP3-associated proteins (ASC, caspase 1) and pro-interleukin-1β (proIL-1β), and phagocytic capacity. RESULTS Monocytes recruited 8 hours after MSU crystal stimulation (F4/80(low) Gr-1(int) 7/4+) exhibited poor phagocytic capacity, expressed low levels of proIL-1β, and failed to produce proinflammatory cytokines in response to MSU crystal restimulation. In the absence of MSU crystal restimulation, differentiating monocytes produced low levels of transforming growth factor β1 ex vivo, and this was abrogated following MSU crystal restimulation. Over time these cells developed a proinflammatory phenotype in vivo, characterized by the production of IL-1β, tumor necrosis factor α, IL-6, CCL2 (monocyte chemotactic protein 1), and CXCL1 (cytokine-induced neutrophil chemoattractant) following ex vivo MSU crystal restimulation, and leading to IL-1β production and cell infiltration following MSU crystal rechallenge in vivo. Proinflammatory function was associated with differentiation toward a macrophage phenotype (F4/80(high) Gr-1-7/4-), an increase in phagocytic capacity, and an increase in the expression of proIL-1β. CONCLUSION MSU crystal-recruited monocytes differentiate into proinflammatory M1-like macrophages in vivo. This proinflammatory macrophage phenotype is likely to play a key role in perpetuating inflammation in gouty arthritis in the presence of ongoing deposition of fresh MSU crystals.

The anti-cancer, anti-inflammatory and tuberculostatic activities of a series of 6,7-substituted-5,8-quinolinequinones

A variety of 6,7-substituted-5,8-quinolinequinones were synthesised and assessed for their anti-tumour and anti-inflammatory activities, and their ability to inhibit the growth of Mycobacterium bovis BCG. In particular, the introduction of a sulfur group at the 7-position of the quinolinequinone led to the discovery of two compounds, 6-methylamino-7-methylsulfanyl-5,8-quinolinequinone (10a) and 6-amino-7-methylsulfonyl-5,8-quinolinequinone (12), that exhibited selectivity for leukemic cells over T-cells, a highly desirable property for an anti-cancer drug. A number of anti-inflammatory (AI) compounds were also identified, with 6,7-bis-methylsulfanyl-5,8-quinolinequinone (18a) exhibiting the highest AI activity (0.11 microM), while 6,7-dichloro-5,8-quinolinequinone (7a), 6,7-dichloro-2-methyl-5,8-quinolinequinone (7b), and 6,7-bis-phenylsulfanyl-quinoline-5,8-diol (19) also exhibited good AI activity and specificity. Several quinolinequinone TB-drug candidates were identified. Of these, 6-amino-7-chloro-5,8-quinolinequinone (11) and 6-amino-7-methanesulfinyl-5,8-quinolinequinone (14), exhibited low MICs (1.56-3.13 microg/mL) for the 100% growth inhibition of M. Bovis BCG. Some general trends pertaining to the functional group substitution of the quinolinequinone core and biological activity were also identified.