Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma.

BACKGROUND: Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality. RESULTS: Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37 × 10(6)-86 × 10(6) unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing. CONCLUSIONS: Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

An outbreak of enterovirus 71 in metropolitan Sydney: enhanced surveillance and lessons learnt.

OBJECTIVE: To report the findings of the enhanced surveillance set up in New South Wales in response to the recent outbreak of human enterovirus 71 (EV71) infection. DESIGN AND SETTING: A two-armed enhanced public health surveillance system including statewide emergency department surveillance and clinical surveillance at the Sydney Children's Hospitals Network. PARTICIPANTS: Children aged less than 10 years with suspected or confirmed enterovirus infection. MAIN OUTCOME MEASURES: Epidemiology of the outbreak, including weekly case counts, demographic information, geographic spread of the outbreak, and clinical presentation and progression. RESULTS: Statewide weekly case counts indicate that an epidemic of EV71 infection occurred in NSW from December 2012 until May 2013. Around 119 children were reported with disease severe enough to warrant admission to a tertiary Sydney children's hospital. Cases were spread throughout the Sydney metropolitan area and there is some evidence of geographic migration of the outbreak. Presenting features included fever, lethargy, myoclonus and skin rash. Only 24% of cases presented with classical hand, foot and mouth disease. CONCLUSIONS: EV71 infection is likely to continue to be a public health problem in Australia. Surveillance of routinely collected emergency department data can provide a useful indication of its activity in the community.

Unexpected protective role for Toll-like receptor 3 in the arterial wall.

The critical role of Toll-like receptors (TLRs) in mammalian host defense has been extensively explored in recent years. The capacity of about 10 TLRs to recognize conserved patterns on many bacterial and viral pathogens is remarkable. With so few receptors, cross-reactivity with self-tissue components often occurs. Previous studies have frequently assigned detrimental roles to TLRs, in particular to TLR2 and TLR4, in immune and cardiovascular disease. Using human and murine systems, we have investigated the consequence of TLR3 signaling in vascular disease. We compared the responses of human atheroma-derived smooth muscle cells (AthSMC) and control aortic smooth muscle cells (AoSMC) to various TLR ligands. AthSMC exhibited a specific increase in TLR3 expression and TLR3-dependent functional responses. Intriguingly, exposure to dsRNA in vitro and in vivo induced increased expression of both pro- and anti-inflammatory genes in vascular cells and tissues. Therefore, we sought to assess the contribution of TLR3 signaling in vivo in mechanical and hypercholesterolemia-induced arterial injury. Surprisingly, neointima formation in a perivascular collar-induced injury model was reduced by the systemic administration of the dsRNA analog Poly(I:C) in a TLR3-dependent manner. Furthermore, genetic deletion of TLR3 dramatically enhanced the development of elastic lamina damage after collar-induced injury. Accordingly, deficiency of TLR3 accelerated the onset of atherosclerosis in hypercholesterolemic ApoE(-/-) mice. Collectively, our data describe a protective role for TLR signaling in the vessel wall.

Smooth muscle cells in human atherosclerosis: proteomic profiling reveals differences in expression of Annexin A1 and mitochondrial proteins in carotid disease.

Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The molecular mechanisms that mediate their properties are incompletely defined. We employed proteomics and in vitro functional assays to identify the unique characteristics of intimal SMC isolated from human carotid endarterectomy specimens and medial SMC from thoracic aortas and carotids. We verified our findings in the Tampere Vascular Study. Human atheroma-derived SMC exhibit decreased expression of mitochondrial proteins ATP Synthase subunit-beta and Aldehyde dehydrogenase 2, and decreased mitochondrial activity when compared to control SMC. Moreover, a comparison between plaque-derived SMC isolated from patients with or without recent acute cerebrovascular symptoms uncovered an increase in Annexin A1, an endogenous anti-inflammatory protein, in the asymptomatic group. The deletion of Annexin A1 or the blockade of its signaling in SMC resulted in increased cytokine production at baseline and after stimulation with the pro-inflammatory cytokine Tumor Necrosis Factor α. In summary, our proteomics and biochemical analysis revealed mitochondrial damage in human plaque-derived SMC as well as a role of Annexin A1 in reducing the production of pro-inflammatory mediators in SMC.

Recombinant tissue plasminogen activator (rt-PA) utilisation by rural clinicians in acute ischaemic stroke: an audit of current practice and clinical outcomes.

OBJECTIVE: This audit of activity reports on current rates of recombinant tissue plasminogen activator (rt-PA) use within specialised stroke care units in rural New South Wales (NSW). It measures stroke onset-to-treatment time and morbidity outcomes for patients treated with rt-PA and aims to establish the safety and effectiveness of rt-PA use in rural NSW. DESIGN, SETTING AND PARTICIPANTS: Medical records reviews of patients admitted with acute ischaemic stroke at two rural NSW hospitals between 1 July 2008 and 30 June 2010. MAIN OUTCOME MEASURES: Treatment with rt-PA, morbidity scores 5 days post-stroke or discharge, incidence of intracranial haemorrhage and mortality rate 6 months post-stroke were recorded. Treatment protocol violations were assessed and time to treatment from stroke onset and hospital admission. RESULTS: Of 605 patients admitted with acute ischaemic stroke, 20 (3.3%) received rt-PA treatment. Of these two, 10% had symptomatic intracranial haemorrhage and one died within 6 months. Morbidity scores for those treated with rt-PA were similar to those not treated. The median onset-to-needle time was 2 hours and 34 min, and the median door-to-needle time was 1 hour and 40 min. There were no treatment protocol violations. CONCLUSION: Recombinant tissue plasminogen activator can be delivered in rural Australian hospitals in a timely manner within recommended implementation guidelines. Acute stroke thrombolytic services in rural Australian facilities had comparable outcomes to metropolitan facilities. Small numbers of thrombolysed patients prevented a validation study of the well-defined outcome benefits from rt-PA. The need for ongoing data collection in regional settings is supported.

Toll-like receptor-2 mediates inflammation and matrix degradation in human atherosclerosis.

BACKGROUND: Inflammation and matrix degradation are the hallmarks of high-risk atherosclerosis that leads to myocardial infarction and stroke. Toll-like receptors (TLRs), key players in innate immunity, are upregulated in atherosclerotic lesions, but their functional role in human atherosclerosis is unknown. We explored the effects of blocking TLR-2, TLR-4, and myeloid differentiation primary response gene 88 (MyD88), a signaling adaptor shared by most TLRs and interleukin-1 receptor (IL-1R), in an in vitro model of human atherosclerosis. METHODS AND RESULTS: Carotid endarterectomies were obtained from patients with symptomatic carotid disease. Cells were isolated via enzymatic tissue dissociation and cultured in the presence or absence of TLR signaling blockers. A dominant-negative form of MyD88 (MyD88(DN)) decreased the production of monocyte chemotactic protein-1/CCL2 (P=0.000), IL-8/CXCL8 (P=0.006), IL-6 (P=0.002), matrix metalloproteinase-1 (MMP-1; P=0.002), and MMP-3 (P=0.000), as well as nuclear factor-kappaB activation (P<0.05) in atheroma cell cultures. IL-1R antagonist, TLR-4 blocking antibodies, or overexpression of a dominant-negative form of the TLR-4 signaling adaptor TRIF-related adaptor molecule reduced nuclear factor-kappaB activity but did not have a broad impact on the production of the mediators studied. In contrast, TLR-2 neutralizing antibodies inhibited nuclear factor-kappaB activation (P<0.05) and significantly reduced monocyte chemotactic protein-1/CCL2 (P=0.000), IL-8/CXCL8 (P=0.009), IL-6 (P=0.000), and MMP-1 (P=0.000), MMP-2 (P=0.004), MMP-3 (P=0.000), and MMP-9 (P=0.006) production. CONCLUSIONS: Our data indicate that TLR-2 signaling through MyD88 plays a predominant role in inflammation and matrix degradation in human atherosclerosis. TLR-2 blockade may represent a therapeutic strategy for atherosclerosis and its complications.