Description: Background— The mechanism whereby the 9p21.3 locus confers risk for coronary artery disease remains incompletely understood. Risk alleles are associated with reduced expression of the cell cycle suppressor genes CDKN2A (p16 and p14) and CDKN2B (p15) and increased vascular smooth muscle cell proliferation. We asked whether risk alleles disrupt transcription factor binding to account for this effect. Methods and Results— A bioinformatic screen was used to predict which of 59 single nucleotide polymorphisms at the 9p21.3 locus disrupt (or create) transcription factor binding sites. Electrophoretic mobility shift and luciferase reporter assays examined the binding and functionality of the predicted regulatory sequences. Primary human aortic smooth muscle cells (HAoSMCs) were genotyped for 9p21.3, and HAoSMCs homozygous for the risk allele showed reduced p15 and p16 levels and increased proliferation. rs10811656 and rs4977757 disrupted functional TEF-1 TEC1 AbaA domain (TEAD) transcription factor binding sites. TEAD3 and TEAD4 overexpression induced p16 in HAoSMCs homozygous for the nonrisk allele, but not for the risk allele. Transforming growth factor β, known to activate p16 and also to interact with TEAD factors, failed to induce p16 or to inhibit proliferation of HAoSMCs homozygous for the risk allele. Knockdown of TEAD3 blocked transforming growth factor β–induced p16 mRNA and protein expression, and dual knockdown of TEAD3 and TEAD4 markedly reduced p16 expression in heterozygous HAoSMCs. Conclusions— Here, we identify a novel mechanism whereby sequences at the 9p21.3 risk locus disrupt TEAD factor binding and TEAD3-dependent transforming growth factor β induction of p16 in HAoSMCs. This mechanism accounts, in part, for the 9p21.3 coronary artery disease risk.

Description: Purpose: To determine whether serum levels of high mobility group box protein 1 (HMGB1) could differentiate malignant mesothelioma patients, asbestos-exposed individuals, and unexposed controls. Experimental Design: Hyperacetylated and nonacetylated HMGB1 (together referred to as total HMGB1) were blindly measured in blood collected from malignant mesothelioma patients ( n = 22), individuals with verified chronic asbestos exposure ( n = 20), patients with benign pleural effusions ( n = 13) or malignant pleural effusions not due to malignant mesothelioma ( n = 25), and healthy controls ( n = 20). Blood levels of previously proposed malignant mesothelioma biomarkers fibulin-3, mesothelin, and osteopontin were also measured in nonhealthy individuals. Results: HMGB1 serum levels reliably distinguished malignant mesothelioma patients, asbestos-exposed individuals, and unexposed controls. Total HMGB1 was significantly higher in malignant mesothelioma patients and asbestos-exposed individuals compared with healthy controls. Hyperacetylated HMGB1 was significantly higher in malignant mesothelioma patients compared with asbestos-exposed individuals and healthy controls, and did not vary with tumor stage. At the cut-off value of 2.00 ng/mL, the sensitivity and specificity of serum hyperacetylated HMGB1 in differentiating malignant mesothelioma patients from asbestos-exposed individuals and healthy controls was 100%, outperforming other previously proposed biomarkers. Combining HMGB1 and fibulin-3 provided increased sensitivity and specificity in differentiating malignant mesothelioma patients from patients with cytologically benign or malignant non–mesothelioma pleural effusion. Conclusions: Our results are significant and clinically relevant as they provide the first biomarker of asbestos exposure and indicate that hyperacetylated HMGB1 is an accurate biomarker to differentiate malignant mesothelioma patients from individuals occupationally exposed to asbestos and unexposed controls. A trial to independently validate these findings will start soon. Clin Cancer Res; 22(12); 3087–96. ©2016 AACR .

Description: A glassy carbon electrode (GCE) was modified with a fullerene/Multiwalled Carbon Nanotubes (MWCNTs)/Nafion composite and applied to the determination of carbendazim, a fungicide. The voltammetric behavior of the analyte was investigated using Cyclic Voltammetry (CV), on the bare GCE and on the same electrode coated by a thin film of the composite material. The electrode response was more than fourfold important on the modified electrode, due to electrical conductivity of fullerene and MWCNT and to favorable electrostatic interaction between the negatively charged Nafion and the protonated fungicide. A sensitive electroanalytical procedure based on Square Wave Voltammetry (SWV) was then developed to detect the analyte. Under the optimum conditions, a linear relationship was obtained between the peak current and the concentration of carbendazim, in the range from 2.0 × 10−8 mol/L to 3.5 × 10−7 mol/L, leading to a detection limit of 1.7 × 10−8 mol/L and to a quantification limit of 5.57 × 10−8 mol/L. The developed procedure was successfully applied to detect carbendazim upon adsorption by some ferritic soils.

Description: Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2–deficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2–HMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4–MD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness.

Description: When a doctor recommends a new medicine there are questions that a patient would hope to have answered. Will the treatment work for my clinical problem? Are there side effects? How many tablets do I...

Description: Objective Liver fibrosis is associated with significant collagen-I deposition largely produced by activated hepatic stellate cells (HSCs); yet, the link between hepatocyte damage and the HSC profibrogenic response remains unclear. Here we show significant induction of osteopontin (OPN) and high-mobility group box-1 (HMGB1) in liver fibrosis. Since OPN was identified as upstream of HMGB1, we hypothesised that OPN could participate in the pathogenesis of liver fibrosis by increasing HMGB1 to upregulate collagen-I expression. Design and results Patients with long-term hepatitis C virus (HCV) progressing in disease stage displayed enhanced hepatic OPN and HMGB1 immunostaining, which correlated with fibrosis stage, whereas it remained similar in non-progressors. Hepatocyte cytoplasmic OPN and HMGB1 expression was significant while loss of nuclear HMGB1 occurred in patients with HCV-induced fibrosis compared with healthy explants. Well-established liver fibrosis along with marked induction of HMGB1 occurred in CCl 4 -injected Opn Hep transgenic yet it was less in wild type and almost absent in Opn –/– mice. Hmgb1 ablation in hepatocytes ( Hmgb1 Hep ) protected mice from CCl 4 -induced liver fibrosis. Coculture with hepatocytes that secrete OPN plus HMGB1 and challenge with recombinant OPN (rOPN) or HMGB1 (rHMGB1) enhanced collagen-I expression in HSCs, which was blunted by neutralising antibodies (Abs) and by Opn or Hmgb1 ablation. rOPN induced acetylation of HMGB1 in HSCs due to increased NADPH oxidase activity and the associated decrease in histone deacetylases 1/2 leading to upregulation of collagen-I. Last, rHMGB1 signalled via receptor for advanced glycation end-products and activated the PI3K–pAkt1/2/3 pathway to upregulate collagen-I. Conclusions During liver fibrosis, the increase in OPN induces HMGB1, which acts as a downstream alarmin driving collagen-I synthesis in HSCs.

Description: Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1 –/– chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.

Description: Objective To obtain pilot data to evaluate the discriminatory power of biomarkers microRNA-122 (miR-122), high-mobility group box 1 (HMGB1), full-length keratin-18 (flk-18) and caspase-cleaved keratin-18 (cck-18) in plasma to identify potential biliary complications that may require acute intervention. Design An observational biomarker cohort pilot study. Setting In a Scottish University teaching hospital for 12 months beginning on 3 September 2014. Participants Blood samples were collected from adults (≥16 years old) referred with acute biliary-type symptoms who have presented to hospital within 24 hours prior were recruited. Patients unable or refused to give informed consent or were transferred from a hospital outside the National Health Service regional trust were excluded. Primary outcome measures To evaluate whether circulating miR-122, HMGB1, flk-18 and cck-18 can discriminate between people with and without gallstone disease and uncomplicated from complicated gallstone disease during the first 24 hours of hospital admission. Results 300 patients were screened of which 285 patients were included. Plasma miR-122, cck-18 and flk-18 concentrations were increased in patients with gallstones compared with those without (miR-122: median: 2.89 x 10 4 copies/mL vs 0.90 x 10 4 copies/mL (p〈0.001); cck-18: 121.2 U/L vs 103.5 U/L (p=0.031); flk-18: 252.4 U/L vs 145.1 U/L (p〈0.001)). Uncomplicated gallstone disease was associated with higher miR-122 and cck-18 concentrations than complicated disease (miR-122: 5.72 x 10 4 copies/mL vs 2.26 x 10 4 copies/mL (p=0.023); cck-18: 139.7 U/L vs 113.6 U/L (p=0.047)). There was no significant difference in HMGB1 concentration between patients with and without gallstones (p=0.559). Separation between groups for all biomarkers was modest. Conclusion miR-122 and keratin-18 plasma concentrations are elevated in patients with gallstones. However, this result is confounded by the association between biomarker concentrations, age and gender. In this pilot study, miR-122 and keratin-18 were not sufficiently discriminatory to be progressed as clinically useful biomarkers in this context.