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The use of non-invasive imaging to identify ruptured or high-risk coronary atherosclerotic plaques would represent a major clinical advance for prevention and treatment of coronary artery disease. We used combined PET and CT to identify ruptured and high-risk atherosclerotic plaques using the radioactive tracers 18F-sodium fluoride (18F-NaF) and 18F-fluorodeoxyglucose (18F-FDG). In this prospective clinical trial, patients with myocardial infarction (n=40) and stable angina (n=40) underwent 18F-NaF and 18F-FDG PET-CT, and invasive coronary angiography. 18F-NaF uptake was compared with histology in carotid endarterectomy specimens from patients with symptomatic carotid disease, and with intravascular ultrasound in patients with stable angina. The primary endpoint was the comparison of 18F-fluoride tissue-to-background ratios of culprit and non-culprit coronary plaques of patients with acute myocardial infarction. In 37 (93%) patients with myocardial infarction, the highest coronary 18F-NaF uptake was seen in the culprit plaque (median maximum tissue-to-background ratio: culprit 1·66 [IQR 1·40–2·25] vs highest non-culprit 1·24 [1·06–1·38], p<0·0001). By contrast, coronary 18F-FDG uptake was commonly obscured by myocardial uptake and where discernible, there were no differences between culprit and non-culprit plaques (1·71 [1·40–2·13] vs 1·58 [1·28–2·01], p=0·34). Marked 18F-NaF uptake occurred at the site of all carotid plaque ruptures and was associated with histological evidence of active calcification, macrophage infiltration, apoptosis, and necrosis. 18 (45%) patients with stable angina had plaques with focal 18F-NaF uptake (maximum tissue-to-background ratio 1·90 [IQR 1·61–2·17]) that were associated with more high-risk features on intravascular ultrasound than those without uptake: positive remodelling (remodelling index 1·12 [1·09–1·19] vs 1·01 [0·94–1·06]; p=0·0004), microcalcification (73% vs 21%, p=0·002), and necrotic core (25% [21–29] vs 18% [14–22], p=0·001). 18F-NaF PET-CT is the first non-invasive imaging method to identify and localise ruptured and high-risk coronary plaque. Future studies are needed to establish whether this method can improve the management and treatment of patients with coronary artery disease. Chief Scientist Office Scotland and British Heart Foundation.

Intracellular protein complexes containing nucleic acids are common targets of autoantibodies in many autoimmune diseases. Central tolerance to these antigens is incomplete, yet nucleosomal DNA is expressed on the surface of cells dying by apoptosis. It is commonly believed that autoimmunity is prevented by the rapid uptake of apoptotic cells (ACs) by neighbors or professional phagocytes to which they deliver anti-inflammatory signals. Self-reactive, innate-like B cells contact and are selected by intracellular antigens expressed on ACs; however, how self-tolerance is maintained is not well understood. Here we report that IL-10 production by B cells, stimulated by contact with ACs, results from the engagement of Toll-like receptor 9 (TLR9) within the B cell after recognition of DNA-containing complexes on the surface of ACs. Until now, TLR9 ligation has been considered an inflammatory signal, but we have confirmed a hitherto unexpected immunoregulatory role by demonstrating the absence of the protective effect of ACs during experimental autoimmune encephalitis (EAE) in TLR9-deficient mice. Human circulating CD27+ B cells also respond to DNA-bearing ACs, but not to DNase-treated cells, by secreting IL-10. Chronic autoimmune disease may arise if this tolerance mechanism is not reimposed after episodes of inflammation, or if the regulatory B-cell response is subverted.

A disintegrin and metalloproteinase 12 (ADAM12) is known to be involved in chondrocyte proliferation and maturation; however, the mechanisms are not fully understood. In this study, expression and localization of ADAM12 during chondrocyte differentiation were examined in the mouse growth plate by immunohistochemistry. Adam12 expression during ATDC5 chondrogenic differentiation was examined by real-time PCR and compared with the expression pattern of type X collagen. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was used to generate Adam12-knockout (KO) ATDC5 cells. Adam12-KO and Adam12 overexpressing cells were used for analyses of ADAM12 expression with or without TGF-β1 stimulation. ADAM12 was identified predominantly in chondrocytes of the proliferative zone in mouse growth plates by immunohistochemistry. Adam12 was upregulated prior to Col10a1 during chondrogenic differentiation in wild-type ATDC5 cells. In Adam12-KO ATDC5 cells, following initiation of chondrogenic differentiation, we observed a reduction in Igf-1 expression along with an upregulation of hypertrophy-associated Runx2, Col10a1, and type X collagen protein expressions. In ATDC5 wild-type cells, stimulation with TGF-β1 upregulated the expressions of Adam12 and Igf-1 and downregulated the expression of Runx2. In contrast, in Adam12-KO ATDC5 cells, these TGF-β1-induced changes were suppressed. Adam12 overexpression resulted in an upregulation of Igf-1 and downregulation of Runx2 expression in ATDC5 cells. The findings suggest that ADAM12 has important role in the regulation of chondrocyte differentiation, potentially by regulation of TGF-β1-dependent signaling and that targeting of ADAM12 may have a role in management of abnormal chondrocyte differentiation.

Undifferentiated pleomorphic sarcoma (UPS) is a rare cancer with limited systemic treatment options and poor outcomes. To seek novel therapeutic interventions, we undertook mutational analysis of 20 UPS patient tumours, four established UPS cell lines and three patient‐derived xenograft (PDX) models. Frequently mutated genes were uncommon; in contrast, copy number (CN) events were common with CN gain frequently observed at genes including JUN, EGFR and CDK6 and loss at WNT8B, RB1 and PTEN. Analysis of overlapping genomic changes between patient tumours and PDX models or cell lines revealed druggable events. A selected panel of drugs targeting these was analysed in in vitro UPS models demonstrating that the mitogen‐activated protein kinase kinase (MEK) inhibitor trametinib is synergistic in combination with the fibroblast growth factor receptor (FGFR) inhibitor infigratinib. This was further confirmed to be efficacious in an ex vivo tumour slice model. Taken together, our results demonstrate the rationale for utilising genomic data to identify drug classes targeting druggable events in low‐prevalence cancers and indicate that trametinib alone or in combination with infigratinib should be further explored for clinical UPS management. GA text Genomic data from undifferentiated pleomorphic sarcoma patients and preclinical models were used to inform a targeted drug screen. Selected compounds were tested in 2D and 3D cultures of UPS cell lines. A combination of trametinib and infigratinib was synergistic in the majority of UPS cell lines tested, which was further confirmed in an ex vivo tumour slice model.

The magnetization of mesenchymal stem cells (MSC) has the potential to aid tissue engineering approaches by allowing tracking, targeting, and local retention of cells at the site of tissue damage. Commonly used methods for magnetizing cells include optimizing uptake and retention of superparamagnetic iron oxide nanoparticles (SPIONs). These appear to have minimal detrimental effects on the use of MSC function as assessed by in vitro assays. The cellular content of magnetic nanoparticles (MNPs) will, however, decrease with cell proliferation and the longer-term effects on MSC function are not entirely clear. An alternative approach to magnetizing MSCs involves genetic modification by transfection with one or more genes derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesizes single-magnetic domain crystals which are incorporated into magnetosomes. MSCs with either or mms6 and mmsF genes are followed by bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by magnetic resonance (MR) and which have no deleterious effects on MSC proliferation, migration, or differentiation. The stable transfection of magnetosome-associated genes in MSCs promotes assimilation of magnetic nanoparticle synthesis into mammalian cells with the potential to allow MR-based cell tracking and, through external or internal magnetic targeting approaches, enhanced site-specific retention of cells for tissue engineering.

Paget's disease of bone (PDB) is characterized by focal increases in bone remodelling. Genome-wide association studies identified a susceptibility locus for PDB tagged by rs5742915, which is located within the PML gene. Here, we have assessed the candidacy of PML as the predisposing gene for PDB at this locus. We found that the PDB-risk allele of rs5742915 was associated with lower PML expression and that PML expression in blood cells from individuals with PDB was lower than in controls. The differentiation, survival and resorptive activity of osteoclasts prepared from Pml−/− mice was increased compared with wild type. Furthermore, the inhibitory effect of IFN-γ on osteoclast formation from Pml−/− was significantly blunted compared with wild type. Bone nodule formation was also increased in osteoblasts from Pml−/− mice when compared with wild type. Although microCT analysis of trabecular bone showed no differences between Pml−/− mice and wild type, bone histomorphometry showed that Pml−/− mice had high bone turnover with increased indices of bone resorption and increased mineral apposition rate. These data indicate that reduced expression of PML predisposes an individual to PDB and identify PML as a novel regulator of bone metabolism. This article has an associated First Person interview with the first author of the paper.

The aim of the study was to investigate the use of a hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Eighteen skeletally mature sheep were treated. The animals were divided into three groups: cell-free scaffold, scaffold seeded with autologous chondrocytes, and meniscectomy alone. The implant was sutured to the capsule and to the meniscal ligament. At a 12-month gross assessment, histology and histomorphometry were used to assess the meniscus implant, knee joint, and osteoarthritis development. All implants showed excellent capsular ingrowth at the periphery. The implant gross assessment showed significant differences between cell-seeded and cell-free groups ( p =0.011). The histological analysis indicated a cellular colonization throughout the implanted constructs. Avascular cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. Joint gross assessment showed that sheep treated with scaffold implantation achieved a significant higher score than those underwent meniscectomy ( p <0.0005), and the Osteoarthritis Research Society International score showed that osteoarthritic changes were significantly less in the cell-seeded group than in the meniscectomy group ( p =0.047), even though results were not significantly superior to those of the cell-free scaffold. Seeding of the scaffold with autologous chondrocytes increases its tissue regeneration capacity, providing a better fibrocartilaginous tissue formation. The study suggests the potential of the novel hyaluronic acid/polycaprolactone scaffold for total meniscal substitution, although this approach has to be further improved before being applied into clinical practice.

Carbon tetrachloride (CCl4) is widely used as an animal model of hepatotoxicity and the mechanisms have been arduously studied, however, the contribution of the sympathetic nervous system (SNS) in CCl4-induced acute hepatotoxicity remains controversial. It is also known that either CCl4 or SNS can affect systemic inflammatory responses. The aim of this study was to establish the effect of chemical sympathectomy with 6-hydroxydopamine (6-OHDA) in a mouse model of CCl4-induced acute hepatotoxicity and systemic inflammatory response. Mice exposed to CCl4 or vehicle were pretreated with 6-OHDA or saline. The serum levels of aminotransferases and alkaline phosphatase in the CCl4-poisoning mice with sympathetic denervation were significantly lower than those without sympathetic denervation. With sympathetic denervation, hepatocellular necrosis and fat infiltration induced by CCl4 were greatly decreased. Sympathetic denervation significantly attenuated CCl4-induced lipid peroxidation in liver and serum. Acute CCl4 intoxication showed increased expression of inflammatory cytokines/chemokines [eotaxin-2/CCL24, Fas ligand, interleukin (IL)-1α, IL-6, IL-12p40p70, monocyte chemoattractant protein-1 (MCP-1/CCL2), and tumor necrosis factor-α (TNF-α)], as well as decreased expression of granulocyte colony-stimulating factor and keratinocyte-derived chemokine. The overexpressed levels of IL-1α, IL-6, IL-12p40p70, MCP-1/CCL2, and TNF-α were attenuated by sympathetic denervation. Pretreatment with dexamethasone significantly reduced CCl4-induced hepatic injury. Collectively, this study demonstrates that the SNS plays an important role in CCl4-induced acute hepatotoxicity and systemic inflammation and the effect may be connected with chemical- or drug-induced hepatotoxicity and circulating immune response.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila . To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened ( Smo ), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

The use of stem cells to support tissue repair is facilitated by loading of the therapeutic cells with magnetic nanoparticles (MNPs) enabling magnetic tracking and targeting. Current methods for magnetizing cells use artificial MNPs and have disadvantages of variable uptake, cellular cytotoxicity and loss of nanoparticles on cell division. Here we demonstrate a transgenic approach to magnetize human mesenchymal stem cells (MSCs). MSCs are genetically modified by transfection with the mms6 gene derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesises single-magnetic domain crystals which are incorporated into magnetosomes. Following transfection of MSCs with the mms6 gene there is bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by MR and which have no deleterious effects on cell proliferation, migration or differentiation. The assimilation of magnetic nanoparticle synthesis into mammalian cells creates a real and compelling, cytocompatible, alternative to exogenous administration of MNPs.