Targeting NETs using dual-active DNase1 variants

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states

Facets of individual-specific health signatures determined from longitudinal plasma proteome profiling

Background: Precision medicine approaches aim to tackle diseases on an individual level through molecular profiling. Despite the growing knowledge about diseases and the reported diversity of molecular phenotypes, the descriptions of human health on an individual level have been far less elaborate. Methods: To provide insights into the longitudinal protein signatures of well-being, we profiled blood plasma collected over one year from 101 clinically healthy individuals using multiplexed antibody assays. After applying an antibody validation scheme, we utilized > 700 protein profiles for in-depth analyses of the individuals’ short-term health trajectories. <p<Findings: We found signatures of circulating proteomes to be highly individual-specific. Considering technical and longitudinal variability, we observed that 49% of the protein profiles were stable over one year. We also identified eight networks of proteins in which 11 242 proteins covaried over time. For each participant, there were unique protein profiles of which some could be explained by associations to genetic variants. Interpretation: This observational and non-interventional study identifyed noticeable diversity among clinically healthy subjects, and facets of individual-specific signatures emerged by monitoring the variability of the circulating proteomes over time. To enable more personal hence precise assessments of health states, longitudinal profiling of circulating proteomes can provide a valuable component for precision medicine approaches

Phenotypic Modulation of Smooth Muscle Cells in Atherosclerosis Is Associated With Downregulation of LMOD1, SYNPO2, PDLIM7, PLN, and SYNM

OBJECTIVE: Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. APPROACH AND RESULTS: Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7, and PLN expression positively correlated to typical SMC markers in plaques (Pearson r>0.6, P0.8, P<0.0001). By immunohistochemistry, the proteins were expressed in SMCs in normal vessels, but largely absent in human plaques and intimal hyperplasia. Subcellularly, most proteins localized to the cytoskeleton in cultured SMCs and were regulated by active enhancer histone modification H3K27ac by chromatin immunoprecipitation-sequencing. Functionally, the genes were downregulated by PDGFB (platelet-derived growth factor beta) and IFNg (interferron gamma), exposure to shear flow stress, and oxLDL (oxidized low-density lipoprotein) loading. Genetic variants in PDLIM7, PLN, and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. CONCLUSIONS: We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation

Core mutations, IL28B polymorphisms and response to peginterferon/ribavirin treatment in Swedish patients with hepatitis C virus genotype 1 infection

<p>Abstract</p> <p>Background</p> <p>Patients infected with hepatitis C virus (HCV) genotype 1 respond poorly to standard treatment with 50% or less achieving sustained virologic response. Predicting outcome is essential and could help avoid unnecessary treatment and reduce health cost. Recently, an association of amino acid substitutions in the core region and treatment outcome was observed in Japanese patients. In the present study, the impact of these mutations on response kinetics and treatment outcome was explored in Caucasian patients.</p> <p>Methods</p> <p>The core region of HCV pre-treatment samples obtained from 50 patients treated with peginterferon/ribavirin in a previous Swedish clinical trial with genotype 1 infection were sequenced. The alleles at rs12979860, a single nucleotide polymorphism (SNP), were assessed in order to identify any co-association with this strong response predictor.</p> <p>Results</p> <p>No association between treatment response and substitutions of core residue 91 was found. In contrast, substitutions of core residue 70 were observed in 6/21 (29%) non-responders, but only in one of 29 responders (p = 0.03), and were more common in subgenotype 1b (R70Q in 6 of 13 strains) than in 1a (R70P in 1 of 37 strains, p = 0.004). The rs12979860 SNP upstream of the IL28B gene was overall the strongest response predictor (p = 0.0001). Core 70 substitutions were associated with poorer response kinetics in patients carrying the CT genotype at rs12979860.</p> <p>Conclusions</p> <p>The results indicate that substitutions of core residue 70 are related to treatment response in Caucasian patients with HCV-1b infection, but are of less importance than IL28B polymorphism.</p

MHC-class-II are expressed in a subpopulation of human neural stem cells in vitro in an IFN gamma-independent fashion and during development

This work was supported by grants from Great Ormond Street Hospital Children’s Charity, Newlife Foundation, the Antony Nolan Trust, a studentship to CAG from Consejo Nacional de Ciencia y Tecnologia (CONACyT) and Instituto Jaliscience de la Juventud (IJJ), Mexico and GOSH NIHR Biomedical Research Centre. The human embryonic and fetal material was provided by the Human Developmental Biology Resource (http://hdbr.org) jointly funded by the Medical Research Council (grant G070089) and The Wellcome Trust (grant GR082557)

The Asp298 allele of endothelial nitric oxide synthase is a risk factor for myocardial infarction among patients with type 2 diabetes mellitus

Background: Endothelial dysfunction plays a central role in atherosclerotic progression and cardiovascular complications of type 2 diabetes mellitus (T2DM). Given the role of nitric oxide in the vascular system, we aimed to test hypotheses of synergy between the common endothelial nitric oxide synthase (eNOS) Asp(298) allele and T2DM in predisposing to acute myocardial infarction (AMI). Methods: In a population-based patient survey with 403 persons with T2DM and 799 healthy subjects from the population without diabetes or hypertension, we analysed the relation between T2DM, sex and the eNOS Asp(298) allele versus the risk for AMI. Results: In an overall analysis, T2DM was a significant independent risk factor for AMI. In patients with T2DM, homozygosity for the eNOS Asp(298) allele was a significant risk factor (HR 3.12 [1.49-6.56], p = 0.003), but not in subjects without diabetes or hypertension. Compared to wild-type non-diabetic subjects, all patients with T2DM had a significantly increased risk of AMI regardless of genotype. This risk was however markedly higher in patients with T2DM homozygous for the Asp(298) allele (HR 7.20 [3.01-17.20], p < 0.001), independent of sex, BMI, systolic blood pressure, serum triglycerides, HDL -cholesterol, current smoking, and leisure time physical activity. The pattern seemed stronger in women than in men. Conclusion: We show here a strong independent association between eNOS genotype and AMI in patients with T2DM. This suggests a synergistic effect of the eNOS Asp(298) allele and diabetes, and confirms the role of eNOS as an important pathological bottleneck for cardiovascular disease in patients with T2DM

Astaxanthin vs placebo on arterial stiffness, oxidative stress and inflammation in renal transplant patients (Xanthin): a randomised controlled trial

Background: There is evidence that renal transplant recipients have accelerated atherosclerosis manifest by increased cardiovascular morbidity and mortality. The high incidence of atherosclerosis is, in part, related to increased arterial stiffness, vascular dysfunction, elevated oxidative stress and inflammation associated with immunosuppressive therapy. The dietary supplement astaxanthin has shown promise as an antioxidant and anti-inflammatory therapeutic agent in cardiovascular disease. The aim of this trial is to investigate the effects of astaxanthin supplementation on arterial stiffness, oxidative stress and inflammation in renal transplant patients

Astaxanthin: A Potential Therapeutic Agent in Cardiovascular Disease

Astaxanthin is a xanthophyll carotenoid present in microalgae, fungi, complex plants, seafood, flamingos and quail. It is an antioxidant with anti-inflammatory properties and as such has potential as a therapeutic agent in atherosclerotic cardiovascular disease. Synthetic forms of astaxanthin have been manufactured. The safety, bioavailability and effects of astaxanthin on oxidative stress and inflammation that have relevance to the pathophysiology of atherosclerotic cardiovascular disease, have been assessed in a small number of clinical studies. No adverse events have been reported and there is evidence of a reduction in biomarkers of oxidative stress and inflammation with astaxanthin administration. Experimental studies in several species using an ischaemia-reperfusion myocardial model demonstrated that astaxanthin protects the myocardium when administered both orally or intravenously prior to the induction of the ischaemic event. At this stage we do not know whether astaxanthin is of benefit when administered after a cardiovascular event and no clinical cardiovascular studies in humans have been completed and/or reported. Cardiovascular clinical trials are warranted based on the physicochemical and antioxidant properties, the safety profile and preliminary experimental cardiovascular studies of astaxanthin

Epstein-Barr Virus Evades CD4+ T Cell Responses in Lytic Cycle through BZLF1-mediated Downregulation of CD74 and the Cooperation of vBcl-2

Evasion of immune T cell responses is crucial for viruses to establish persistence in the infected host. Immune evasion mechanisms of Epstein-Barr virus (EBV) in the context of MHC-I antigen presentation have been well studied. In contrast, viral interference with MHC-II antigen presentation is less well understood, not only for EBV but also for other persistent viruses. Here we show that the EBV encoded BZLF1 can interfere with recognition by immune CD4+ effector T cells. This impaired T cell recognition occurred in the absence of a reduction in the expression of surface MHC-II, but correlated with a marked downregulation of surface CD74 on the target cells. Furthermore, impaired CD4+ T cell recognition was also observed with target cells where CD74 expression was downregulated by shRNA-mediated inhibition. BZLF1 downregulated surface CD74 via a post-transcriptional mechanism distinct from its previously reported effect on the CIITA promoter. In addition to being a chaperone for MHC-II αβ dimers, CD74 also functions as a surface receptor for macrophage Migration Inhibitory Factor and enhances cell survival through transcriptional upregulation of Bcl-2 family members. The immune-evasion function of BZLF1 therefore comes at a cost of induced toxicity. However, during EBV lytic cycle induced by BZLF1 expression, this toxicity can be overcome by expression of the vBcl-2, BHRF1, at an early stage of lytic infection. We conclude that by inhibiting apoptosis, the vBcl-2 not only maintains cell viability to allow sufficient time for synthesis and accumulation of infectious virus progeny, but also enables BZLF1 to effect its immune evasion function