In vitro modelling of disease-induced changes in the diabetic wound fibroblast.

OBJECTIVE: Fibroblasts have been shown to play an increasingly important role within diabetic wounds. While several in vitro models of diabetic wound fibroblasts have been reported, none replicate the natural progression of the disease over time, recapitulating the acquisition of the diseased phenotype. Therefore, this study aimed to establish an in vitro model of the diabetic wound fibroblast through sustained exposure of healthy dermal fibroblasts to hyperglycaemia. METHOD: Primary human fibroblasts were isolated from discarded healthy skin tissue and were either exposed to normoglycaemic (control 5.5mM glucose) media or hyperglycaemic (25mM glucose) media for four weeks. Quantitative polymerase chain reaction was performed to measure the expression of inflammatory cytokines and chemokines. RESULTS: In the hyperglycaemia model, stromal cell-derived factor (SDF)-1 expression remained consistently downregulated across all four weeks (p<0.01), while monocyte chemoattractant protein (MCP)-1 (p<0.001), interleukin (IL)-8 (p=0.847) and chemokine (C-X-C motif) ligand 1 (CXCL1) (p=0.872) were initially downregulated at one week followed by subsequent upregulation between 2-4 weeks. CONCLUSION: This hyperglycaemia model may serve as a useful tool to characterise pathological changes in the diabetic wound fibroblast and help identify candidate therapeutic targets, such as SDF-1, that may reverse the pathology.

Bioactive glasses and electrospun composites that release cobalt to stimulate the HIF pathway for wound healing applications.

BACKGROUND: Bioactive glasses are traditionally associated with bonding to bone through a hydroxycarbonate apatite (HCA) surface layer but the release of active ions is more important for bone regeneration. They are now being used to deliver ions for soft tissue applications, particularly wound healing. Cobalt is known to simulate hypoxia and provoke angiogenesis. The aim here was to develop new bioactive glass compositions designed to be scaffold materials to locally deliver pro-angiogenic cobalt ions, at a controlled rate, without forming an HCA layer, for wound healing applications. METHODS: New melt-derived bioactive glass compositions were designed that had the same network connectivity (mean number of bridging covalent bonds between silica tetrahedra), and therefore similar biodegradation rate, as the original 45S5 Bioglass. The amount of magnesium and cobalt in the glass was varied, with the aim of reducing or removing calcium and phosphate from the compositions. Electrospun poly(ε-caprolactone)/bioactive glass composites were also produced. Glasses were tested for ion release in dissolution studies and their influence on Hypoxia-Inducible Factor 1-alpha (HIF-1α) and expression of Vascular Endothelial Growth Factor (VEGF) from fibroblast cells was investigated. RESULTS: Dissolution tests showed the silica rich layer differed depending on the amount of MgO in the glass, which influenced the delivery of cobalt. The electrospun composites delivered a more sustained ion release relative to glass particles alone. Exposing fibroblasts to conditioned media from these composites did not cause a detrimental effect on metabolic activity but glasses containing cobalt did stabilise HIF-1α and provoked a significantly higher expression of VEGF (not seen in Co-free controls). CONCLUSIONS: The composite fibres containing new bioactive glass compositions delivered cobalt ions at a sustained rate, which could be mediated by the magnesium content of the glass. The dissolution products stabilised HIF-1α and provoked a significantly higher expression of VEGF, suggesting the composites activated the HIF pathway to stimulate angiogenesis.

Simple isolation method for the bulk isolation of wear particles from metal on metal bearing surfaces generated in a hip simulator test.

Isolation and characterization of metal-on-metal (MoM) wear particles from simulator lubricants is essential to understand wear behaviour, ion release and associated corrosive activity related to the wear particles. Substantial challenges remain to establish a simple, precise and repeatable protocol for the isolation and analysis of wear particles due to their extremely small size, their tendency to agglomerate and degrade. In this paper, we describe a simple and efficient method for the bulk isolation and characterisation of wear particles from MoM bearings. Freeze drying was used to remove the large volume of water from the serum lubricant, enzymes used to digest the proteins and ultracentrifugation to finally isolate and purify the particles. The present study involved a total of eight steps for the isolation process and a wear particle extraction efficiency of 45% was achieved.

Mycolactone inhibits monocyte cytokine production by a posttranscriptional mechanism.

The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1beta, IL-6, IL-10, and IFN-gamma-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by mycolactone, irrespective of the stimulating ligand. However, mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-kappaB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.

A late, prolonged activation of the phosphatidylinositol 3-kinase pathway is required for T cell proliferation.

Activation of the phosphatidylinositol-3 kinase (PI 3-K) pathway is associated with the proliferation of many cell types, including T lymphocytes. However, recent studies in cell lines stably expressing deletion mutants of IL-2R that fail to activate PI 3-K have questioned the requirement for this pathway in cell cycle regulation. In this study with IL-2 and IL-7, we show in primary T cells that, unlike IL-2, IL-7 fails to induce the early activation of PI 3-K seen within minutes and normally associated with cytokine signaling. However, kinetic experiments showed that both of these T cell growth factors induce a distinct and sustained phase of PI 3-K activity several hours after stimulation. This delayed activation correlates with cell cycle induction and from studies using inhibitors of PI 3-K signaling, we show that this later phase, unlike the early activation within minutes, is required for cell cycle induction. The data presented here will have major implications for our understanding of the mechanism of T cell proliferation as well as the regulation of PI 3-K activity.

IL-4 regulation of p38 MAPK signalling is dependent on cell type.

p38 MAPK was originally characterized as a stress-induced kinase, along with JNK. Subsequently, p38 MAPK was found to be activated by stimuli other than cellular stress, such as growth factors and mitogens, like interleukin (IL)-2, IL-7 and IL-3. A notable exception was IL-4, as studies in mast cells showed no activation of p38 MAPK by this cytokine. In this study we show that the regulation of p38 MAPK is cell type dependent. Like other cytokines that signal through the gamma (gamma)(c), IL-4 can activate p38 MAPK in the CT6 T-cell line and BA/F3 pro-B-cells. However, IL-4 was unable to activate p38 MAPK in the murine macrophage cell line, RAW 264.7 and, indeed, prolonged exposure of cells to IL-4 results in suppression of LPS-induced MAPK activation. This result correlates with the well defined inhibitory effect of IL-4 on tumour necrosis factor alpha (TNFalpha) production. In contrast, studies in primary human monocytes showed that prolonged exposure to IL-4 resulted in enhanced activation of LPS-stimulated p38 MAPK; this correlated with an enhanced TNFalpha production. These data highlight the complexity of IL-4 signalling mechanisms, the diversity that can exist in the regulation of a given signalling pathway by a given cytokine and, furthermore, indicate the problems that can arise from extrapolation between different cell systems.