query: LDL cholesterol has a causal role in the development of cardiovascular disease.

title: LDL-cholesterol concentrations: a genome-wide association study

text:
BACKGROUND LDL cholesterol has a causal role in the development of cardiovascular disease. Improved understanding of the biological mechanisms that underlie the metabolism and regulation of LDL cholesterol might help to identify novel therapeutic targets. We therefore did a genome-wide association study of LDL-cholesterol concentrations.
 METHODS We used genome-wide association data from up to 11,685 participants with measures of circulating LDL-cholesterol concentrations across five studies, including data for 293 461 autosomal single nucleotide polymorphisms (SNPs) with a minor allele frequency of 5% or more that passed our quality control criteria. We also used data from a second genome-wide array in up to 4337 participants from three of these five studies, with data for 290,140 SNPs. We did replication studies in two independent populations consisting of up to 4979 participants. Statistical approaches, including meta-analysis and linkage disequilibrium plots, were used to refine association signals; we analysed pooled data from all seven populations to determine the effect of each SNP on variations in circulating LDL-cholesterol concentrations.
 FINDINGS In our initial scan, we found two SNPs (rs599839 [p=1.7x10(-15)] and rs4970834 [p=3.0x10(-11)]) that showed genome-wide statistical association with LDL cholesterol at chromosomal locus 1p13.3. The second genome screen found a third statistically associated SNP at the same locus (rs646776 [p=4.3x10(-9)]). Meta-analysis of data from all studies showed an association of SNPs rs599839 (combined p=1.2x10(-33)) and rs646776 (p=4.8x10(-20)) with LDL-cholesterol concentrations. SNPs rs599839 and rs646776 both explained around 1% of the variation in circulating LDL-cholesterol concentrations and were associated with about 15% of an SD change in LDL cholesterol per allele, assuming an SD of 1 mmol/L.   INTERPRETATION We found evidence for a novel locus for LDL cholesterol on chromosome 1p13.3. These results potentially provide insight into the biological mechanisms that underlie the regulation of LDL cholesterol and might help in the discovery of novel therapeutic targets for cardiovascular disease.

title: How to control residual cardiovascular risk despite statin treatment: focusing on HDL-cholesterol.

text: Lowering low-density lipoprotein-cholesterol (LDL-C) is the primary target in the management of dyslipidemia in patients at high risk of cardiovascular disease. However, patients who have achieved LDL-C levels below the currently recommended targets may still experience cardiovascular events. This may result, in part, from elevated triglyceride (TG) levels and low levels of high-density lipoprotein-cholesterol (HDL-C). Low HDL-C and high TG levels are common and are recognized as independent risk factors for cardiovascular morbidity and mortality. Furthermore, atherogenic dyslipidemia, characterized by low levels of HDL-C, high TG, and small, dense LDL particles, is a typical phenotype of dyslipidemia in subjects with insulin resistance and metabolic syndrome. Therefore, to reduce further the risk of coronary heart disease (CHD), raising HDL-C and lowering TG may be the secondary therapeutic target for patients who achieve LDL-C levels below the currently recommended targets but are still at risk of CHD. However, whether increasing HDL-C levels alone reduces CHD has not yet been confirmed in large randomized clinical trials, and whether functional HDL is more important than HDL-C in reducing CHD remains controversial. Large CHD endpoint trials that include many patients with diabetes are underway to compare combination treatments with statin and niacin, fibrates, or cholesteryl ester transfer protein inhibitors with statin alone treatments. In this review, we discuss the rationale and importance of increasing HDL-C levels with and without lowering TG levels in the treatment and prevention of cardiovascular events.

title: MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels

text: The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element–binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease.

title: Cardiovascular risk factors in childhood and carotid artery intima-media thickness in adulthood: the Cardiovascular Risk in Young Finns Study.

text:
CONTEXT Exposure to cardiovascular risk factors during childhood and adolescence may be associated with the development of atherosclerosis later in life.
 OBJECTIVE To study the relationship between cardiovascular risk factors measured in childhood and adolescence and common carotid artery intima-media thickness (IMT), a marker of preclinical atherosclerosis, measured in adulthood.
 DESIGN, SETTING, AND PARTICIPANTS Population-based, prospective cohort study conducted at 5 centers in Finland among 2229 white adults aged 24 to 39 years who were examined in childhood and adolescence at ages 3 to 18 years in 1980 and reexamined 21 years later, between September 2001 and January 2002.
 MAIN OUTCOME MEASURES Association between cardiovascular risk variables (levels of low-density lipoprotein cholesterol [LDL-C], high-density lipoprotein cholesterol [HDL-C], and triglycerides; LDL-C/HDL-C ratio; systolic and diastolic blood pressure; body mass index; smoking) measured in childhood and adulthood and common carotid artery IMT measured in adulthood.
 RESULTS In multivariable models adjusted for age and sex, IMT in adulthood was significantly associated with childhood LDL-C levels (P =.001), systolic blood pressure (P<.001), body mass index (P =.007), and smoking (P =.02), and with adult systolic blood pressure (P<.001), body mass index (P<.001), and smoking (P =.004). The number of risk factors measured in 12- to 18-year-old adolescents, including high levels (ie, extreme age- and sex-specific 80th percentile) of LDL-C, systolic blood pressure, body mass index, and cigarette smoking, were directly related to carotid IMT measured in young adults at ages 33 through 39 years (P<.001 for both men and women), and remained significant after adjustment for contemporaneous risk variables. The number of risk factors measured at ages 3 to 9 years demonstrated a weak direct relationship with carotid IMT at ages 24 to 30 years in men (P =.02) but not in women (P =.63).
 CONCLUSIONS Risk factor profile assessed in 12- to 18-year-old adolescents predicts adult common carotid artery IMT independently of contemporaneous risk factors. These findings suggest that exposure to cardiovascular risk factors early in life may induce changes in arteries that contribute to the development of atherosclerosis.

title: A Century of Cholesterol and Coronaries: From Plaques to Genes to Statins

text: One-fourth of all deaths in industrialized countries result from coronary heart disease. A century of research has revealed the essential causative agent: cholesterol-carrying low-density lipoprotein (LDL). LDL is controlled by specific receptors (LDLRs) in liver that remove it from blood. Mutations that eliminate LDLRs raise LDL and cause heart attacks in childhood, whereas mutations that raise LDLRs reduce LDL and diminish heart attacks. If we are to eliminate coronary disease, lowering LDL should be the primary goal. Effective means to achieve this goal are currently available. The key questions are: who to treat, when to treat, and how long to treat.

title: Apolipoprotein E and atherosclerosis: beyond lipid effect.

text: In humans, apolipoprotein E (apoE) is a polymorphic multifunctional protein.1 It is coded by three alleles (e2, e3, e4) of a modulator gene (level, variability, and susceptibility gene) at the apoE locus on chromosome 19, determining six apoE genotypes and plasma phenotypes. Its pleiotropic effects are exerted on plasma lipoprotein metabolism, coagulation, oxidative processes, macrophage, glial cell and neuronal cell homeostasis, adrenal function, central nervous system physiology, inflammation, and cell proliferation.2,3 ApoE polymorphism modulates susceptibility to many diseases. It is, however, particularly notorious for its role in neurodegenerative disorders4 and atherosclerotic arterial disease.5,6 The e4 allele (phenotypes E4/4 and E4/3) that is associated with higher low density lipoprotein cholesterol (LDL-C) is considered proatherogenic, whereas the presence of the e2 allele (E3/2, E2/2), being associated with lower LDL-C levels, is deemed to have the opposite effect (although it may be associated with increased plasma triglycerides and lipoprotein remnants). This simple equation, however, is an oversimplification because these properties are subject to many environmental and genetic influences. ApoE has allele- and gender-dependent effects on reverse cholesterol transport, platelet aggregation, and oxidative processes that are likely to affect the overall atherogenic potential ascribed to modulation of lipoprotein metabolism.2,3,6 Notwithstanding the context dependency, a recent meta-analysis fully supports the presence of the e4 allele as a significant risk factor for coronary artery disease.7 Several mechanisms have been evoked to link apoE with atherosclerosis, but the relationship is not fully unraveled in humans. Nevertheless, some apoE mimetic peptides that promote LDL clearance are currently tested in animals for potential clinical applications.8,9  See page 436   The situation is relatively simpler in animals. The mouse model has been prominently useful to test mechanisms …

title: Novel Risk Factors for Systemic Atherosclerosis: A Comparison of C-Reactive Protein, Fibrinogen, Homocysteine, Lipoprotein(a), and Standard Cholesterol Screening as Predictors of Peripheral Arterial Disease

text: ContextSeveral novel risk factors for atherosclerosis have recently been proposed, but few comparative data exist to guide clinical use of these emerging biomarkers. ObjectiveTo compare the predictive value of 11 lipid and nonlipid biomarkers as risk factors for development of symptomatic peripheral arterial disease (PAD).Design, Setting, and ParticipantsNested case-control study using plasma samples collected at baseline from a prospective cohort of 14 916 initially healthy US male physicians aged 40 to 84 years, of whom 140 subsequently developed symptomatic PAD (cases); 140 age- and smoking status–matched men who remained free of vascular disease during an average 9-year follow-up period were randomly selected as controls. Main Outcome MeasureIncident PAD, as determined by baseline total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol–HDL-C ratio, triglycerides, homocysteine, C-reactive protein (CRP), lipoprotein(a), fibrinogen, and apolipoproteins (apo) A-I and B-100.ResultsIn univariate analyses, plasma levels of total cholesterol (P<.001), LDL-C (P = .001), triglycerides (P = .001), apo B-100 (P = .001), fibrinogen (P = .02), CRP (P = .006), and the total cholesterol–HDL-C ratio (P<.001) were all significantly higher at baseline among men who subsequently developed PAD compared with those who did not, while levels of HDL-C (P = .009) and apo A-I (P = .05) were lower. Nonsignificant baseline elevations of lipoprotein(a) (P = .40) and homocysteine (P = .90) were observed. In multivariable analyses, the total cholesterol–HDL-C ratio was the strongest lipid predictor of risk (relative risk [RR] for those in the highest vs lowest quartile, 3.9; 95% confidence interval [CI], 1.7-8.6), while CRP was the strongest nonlipid predictor (RR for the highest vs lowest quartile, 2.8; 95% CI, 1.3-5.9). In assessing joint effects, addition of CRP to standard lipid screening significantly improved risk prediction models based on lipid screening alone (P<.001).ConclusionsOf 11 atherothrombotic biomarkers assessed at baseline, the total cholesterol–HDL-C ratio and CRP were the strongest independent predictors of development of peripheral arterial disease. C-reactive protein provided additive prognostic information over standard lipid measures.

title: LDL inhibits the mediation of cholesterol efflux from macrophage foam cells by apoA-I-containing lipoproteins. A putative mechanism for foam cell formation.

text: Although the accumulation of cholesterol in macrophages appears to be an initial step in atherogenesis, low-density lipoprotein (LDL), a major risk factor for atherosclerosis, does not promote cholesterol accumulation in macrophages in its native form. On the other hand, apolipoprotein (apo) A-I-containing lipoprotein removes cholesterol from cholesterol-loaded macrophages (foam cells) and prevents cholesterol from accumulating in the cells. We examined the effect of LDL on cholesterol removal by two species of apoA-I-containing lipoproteins, one containing only apoA-I (LpA-I) and the other containing apoA-I and apoA-II (LpA-I/A-II). When foam cells were incubated with LpA-I or LpA-I/A-II, cellular cholesterol mass was reduced. In contrast, when LDL was added, the cholesterol-reducing capacities of these lipoproteins were dose-dependently inhibited by LDL. In the presence of LDL, LpA-I and LpA-I/A-II removed free cholesterol preferentially from LDL rather than from the plasma membrane of foam cells. In addition, a fair amount of cellular cholesterol was directly moved to LDL rather than to LpA-I or LpA-I/A-II. The cellular cholesterol that moved to LDL was completely compensated for by the cholesterol influx from LDL to foam cells. Thus, net cholesterol efflux (a combination of influx and efflux) from foam cells was inhibited by LDL. These results, taken together, indicate that LDL may accelerate foam cell formation by inhibiting cholesterol removal from the cells and that elevated levels of plasma LDL may become a risk factor for atherosclerosis by inhibiting the function of LpA-I and LpA-I/A-II at the cellular level.

title: Mendelian Randomization Study of B-Type Natriuretic Peptide and Type 2 Diabetes: Evidence of Causal Association from Population Studies

text:
BACKGROUND Genetic and epidemiological evidence suggests an inverse association between B-type natriuretic peptide (BNP) levels in blood and risk of type 2 diabetes (T2D), but the prospective association of BNP with T2D is uncertain, and it is unclear whether the association is confounded.
 METHODS AND FINDINGS We analysed the association between levels of the N-terminal fragment of pro-BNP (NT-pro-BNP) in blood and risk of incident T2D in a prospective case-cohort study and genotyped the variant rs198389 within the BNP locus in three T2D case-control studies. We combined our results with existing data in a meta-analysis of 11 case-control studies. Using a Mendelian randomization approach, we compared the observed association between rs198389 and T2D to that expected from the NT-pro-BNP level to T2D association and the NT-pro-BNP difference per C allele of rs198389. In participants of our case-cohort study who were free of T2D and cardiovascular disease at baseline, we observed a 21% (95% CI 3%-36%) decreased risk of incident T2D per one standard deviation (SD) higher log-transformed NT-pro-BNP levels in analysis adjusted for age, sex, body mass index, systolic blood pressure, smoking, family history of T2D, history of hypertension, and levels of triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. The association between rs198389 and T2D observed in case-control studies (odds ratio = 0.94 per C allele, 95% CI 0.91-0.97) was similar to that expected (0.96, 0.93-0.98) based on the pooled estimate for the log-NT-pro-BNP level to T2D association derived from a meta-analysis of our study and published data (hazard ratio = 0.82 per SD, 0.74-0.90) and the difference in NT-pro-BNP levels (0.22 SD, 0.15-0.29) per C allele of rs198389. No significant associations were observed between the rs198389 genotype and potential confounders.
 CONCLUSIONS Our results provide evidence for a potential causal role of the BNP system in the aetiology of T2D. Further studies are needed to investigate the mechanisms underlying this association and possibilities for preventive interventions. Please see later in the article for the Editors' Summary.

title: Effects of PCSK9 Inhibition With Alirocumab on Lipoprotein Metabolism in Healthy Humans

text:
BACKGROUND Alirocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), lowers plasma low-density lipoprotein (LDL) cholesterol and apolipoprotein B100 (apoB). Although studies in mice and cells have identified increased hepatic LDL receptors as the basis for LDL lowering by PCSK9 inhibitors, there have been no human studies characterizing the effects of PCSK9 inhibitors on lipoprotein metabolism. In particular, it is not known whether inhibition of PCSK9 has any effects on very low-density lipoprotein or intermediate-density lipoprotein (IDL) metabolism. Inhibition of PCSK9 also results in reductions of plasma lipoprotein (a) levels. The regulation of plasma Lp(a) levels, including the role of LDL receptors in the clearance of Lp(a), is poorly defined, and no mechanistic studies of the Lp(a) lowering by alirocumab in humans have been published to date.
 METHODS Eighteen (10 F, 8 mol/L) participants completed a placebo-controlled, 2-period study. They received 2 doses of placebo, 2 weeks apart, followed by 5 doses of 150 mg of alirocumab, 2 weeks apart. At the end of each period, fractional clearance rates (FCRs) and production rates (PRs) of apoB and apo(a) were determined. In 10 participants, postprandial triglycerides and apoB48 levels were measured.
 RESULTS Alirocumab reduced ultracentrifugally isolated LDL-C by 55.1%, LDL-apoB by 56.3%, and plasma Lp(a) by 18.7%. The fall in LDL-apoB was caused by an 80.4% increase in LDL-apoB FCR and a 23.9% reduction in LDL-apoB PR. The latter was due to a 46.1% increase in IDL-apoB FCR coupled with a 27.2% decrease in conversion of IDL to LDL. The FCR of apo(a) tended to increase (24.6%) without any change in apo(a) PR. Alirocumab had no effects on FCRs or PRs of very low-density lipoproteins-apoB and very low-density lipoproteins triglycerides or on postprandial plasma triglycerides or apoB48 concentrations.
 CONCLUSIONS Alirocumab decreased LDL-C and LDL-apoB by increasing IDL- and LDL-apoB FCRs and decreasing LDL-apoB PR. These results are consistent with increases in LDL receptors available to clear IDL and LDL from blood during PCSK9 inhibition. The increase in apo(a) FCR during alirocumab treatment suggests that increased LDL receptors may also play a role in the reduction of plasma Lp(a).    CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01959971.

title: Estimating low-density lipoprotein cholesterol by the Friedewald equation is adequate for classifying patients on the basis of nationally recommended cutpoints.

text: We compared low-density lipoprotein cholesterol (LDL) values obtained by the Friedewald formula--i.e., total cholesterol minus high-density lipoprotein (HDL) cholesterol minus very-low-density lipoprotein (VLDL) cholesterol (estimated as triglyceride divided by 5)--with those obtained by lipoprotein fractionation, using 4736 specimens. When triglycerides were less than 2.0 g/L, greater than 90% of estimated LDL cholesterol values were acceptable, within +/- 10% of measured values. At triglyceride concentrations of 2.0-4.0 g/L and 4.0-6.0 g/L, only 72% and 39%, respectively, of the estimates were acceptable. LDL values derived from an alternative formula, estimating VLDL as triglycerides divided by 6, were even less accurate. Nevertheless, the use of estimated LDL for risk classification based on the National Cholesterol Education Program Adult Treatment Panel cutpoints of 1.30 and 1.60 g/L was considered acceptable. At triglyceride concentrations less than or equal to 5.0 g/L, 88% of classifications based on estimated LDL (using triglycerides divided by 5) were concordant with those by measured LDL. Eleven percent of classifications were shifted across one cutpoint, evenly distributed between high and low. Fewer than 1% of classifications, all with Type III hyperlipoproteinemia, were misclassified two cutpoints high. Refinements in the estimation model did not substantially improve LDL estimation or concordance of risk classification.

title: Effects of FXR in foam-cell formation and atherosclerosis development.

text: Farnesoid X receptor (FXR), a bile-acid-activated member of the nuclear receptor superfamily, is essential in regulating bile-acid, cholesterol, and triglyceride homeostasis. Disruption of the FXR gene in mice results in a proatherosclerotic lipid profile with increased serum cholesterols and triglycerides. However, the role of FXR in foam-cell formation and atherosclerosis development remains unclear. The current study showed that the peritoneal macrophages isolated from FXR-null mice took up less oxidized LDL-cholesterol (oxLDL-C), which was accompanied by a marked reduction in CD36 expression in these cells. This result appears to be FXR-independent, as FXR was not detected in the peritoneal macrophages. To assess to what extent FXR modulates atherosclerosis development, FXR/ApoE double-null mice were generated. Female mice were used for atherosclerosis analysis. Compared to ApoE-null mice, the FXR/ApoE double-null mice were found to have less atherosclerotic lesion area in the aorta, despite a further increase in the serum cholesterols and triglycerides. Our results indicate that disruption of the FXR gene could attenuate atherosclerosis development, most likely resulting from reduced oxLDL-C uptake by macrophages. Our study cautions the use of serum lipid levels as a surrogate marker to determine the efficiency of FXR modulators in treating hyperlipidemia.

title: Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice.

text: Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) have been implicated in atherogenesis. However, the precise role of TNF-alpha in atherogenesis is still unclear. To examine the effect of TNF-alpha on atherogenesis, we generated compound-deficient mice in apolipoprotein E (apoE) and TNF-alpha (apoE-/-/TNF-alpha-/-) and compared them with apoE-/- mice. Although serum total cholesterol levels were markedly elevated in both apoE-/-/TNF-alpha-/- and apoE-/- mice compared to wild-type mice, no differences were observed between apoE-/-/TNF-alpha-/- and apoE-/- mice. The atherosclerotic plaque area in the aortic luminal surface of apoE-/-/TNF-alpha-/- mice (n=8, 3.1+/-0.4%) was significantly smaller than that of apoE-/- mice (n=7, 4.7+/-0.4%, p<0.001) despite the lack of difference in serum cholesterol levels. The atherosclerotic lesion size in the aortic sinus of apoE-/-/TNF-alpha-/- mice (n=10, 5.1+/-0.3 x 10(5)microm(2)) was also significantly smaller than that of apoE-/- mice (n=11, 7.0+/-0.3 x 10(5)microm(2), p<0.0001). RT-PCR analysis indicated that the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in apoE-/- than apoE-/-/TNF-alpha-/- mice. Macrophages from apoE(-/-) mice showed higher uptake level of oxidized LDL and increased expression level of scavenger receptor class A (SRA) compared to those from apoE-/-/TNF-alpha-/- mice. These results indicate that TNF-alpha plays an atherogenic role by upregulating the expressions of ICAM-1, VCAM-1 and MCP-1 in the vascular wall, and by inducing SRA expression and oxidized LDL uptake in macrophages.

title: Longitudinal Genome-Wide Association of Cardiovascular Disease Risk Factors in the Bogalusa Heart Study

text: Cardiovascular disease (CVD) is the leading cause of death worldwide. Recent genome-wide association (GWA) studies have pinpointed many loci associated with CVD risk factors in adults. It is unclear, however, if these loci predict trait levels at all ages, if they are associated with how a trait develops over time, or if they could be used to screen individuals who are pre-symptomatic to provide the opportunity for preventive measures before disease onset. We completed a genome-wide association study on participants in the longitudinal Bogalusa Heart Study (BHS) and have characterized the association between genetic factors and the development of CVD risk factors from childhood to adulthood. We report 7 genome-wide significant associations involving CVD risk factors, two of which have been previously reported. Top regions were tested for replication in the Young Finns Study (YF) and two associations strongly replicated: rs247616 in CETP with HDL levels (combined P = 9.7 x 10(-24)), and rs445925 at APOE with LDL levels (combined P = 8.7 x 10(-19)). We show that SNPs previously identified in adult cross-sectional studies tend to show age-independent effects in the BHS with effect sizes consistent with previous reports. Previously identified variants were associated with adult trait levels above and beyond those seen in childhood; however, variants with time-dependent effects were also promising predictors. This is the first GWA study to evaluate the role of common genetic variants in the development of CVD risk factors in children as they advance through adulthood and highlights the utility of using longitudinal studies to identify genetic predictors of adult traits in children.

title: Metabolomics study of the therapeutic mechanism of Schisandra Chinensis lignans in diet-induced hyperlipidemia mice

text:
BACKGROUND Schisandra, a globally distributed plant, has been widely applied for the treatment of diseases such as hyperlipidemia, fatty liver and obesity in China. In the present work, a rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q-TOF-MS)-based metabolomics was conducted to investigate the intervention effect of Schisandra chinensis lignans (SCL) on hyperlipidemia mice induced by high-fat diet (HFD).
 METHODS Hyperlipidemia mice were orally administered with SCL (100 mg/kg) once a day for 4 weeks. Serum biochemistry assay of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) was conducted to confirm the treatment of SCL on lipid regulation. Metabolomics analysis on serum samples was carried out, and principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were carried out for the pattern recognition and characteristic metabolites identification. The relative levels of critical regulatory factors of liver lipid metabolism, sterol regulatory element-binding proteins (SREBPs) and its related gene expressions were measured by quantitative real-time polymerase chain reaction (RT-PCR) for investigating the underlying mechanism.
 RESULTS Oral administration of SCL significantly decreased the serum levels of TC, TG and LDL-c and increased the serum level of HDL-c in the hyperlipidemia mice, and no effect of SCL on blood lipid levels was observed in control mice. Serum samples were scattered in the PCA scores plots in response to the control, HFD and SCL group. Totally, thirteen biomarkers were identified and nine of them were recovered to the normal levels after SCL treatment. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, the anti-hyperlipidemia mechanisms of SCL may be involved in the following metabolic pathways: tricarboxylic acid (TCA) cycle, synthesis of ketone body and cholesterol, choline metabolism and fatty acid metabolism. Meanwhile, SCL significantly inhibited the mRNA expression level of hepatic lipogenesis genes such as SREBP-1c, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), and decreased the mRNA expression of liver X receptor α (LXRα). Moreover, SCL also significantly decreased the expression level of SREBP-2 and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) in the liver of hyperlipidemia mice.
 CONCLUSION Anti-hyperlipidemia effect of SCL was confirmed by both serum biochemistry and metabolomics analysis. The mechanism may be related to the down-regulation of LXRα/SREBP-1c/FAS/ACC and SREBP2/HMGCR signaling pathways.

title: Effects of soy isoflavones and phytate on homocysteine, C-reactive protein, and iron status in postmenopausal women.

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BACKGROUND Soy protein or its components may protect against the atherosclerotic cardiovascular disease (CVD) risk factors total homocysteine (tHcy), C-reactive protein (CRP), and excess body iron, which generally increase with menopause.
 OBJECTIVE The primary objective of this study was to determine the independent effect of the soy protein components isoflavones and phytate on CVD risk factors in postmenopausal women. The secondary objective was to identify factors [blood lipids, oxidative stress indexes, serum ferritin, plasma folate, plasma vitamin B-12, and body mass index (BMI)] contributing to tHcy and CRP concentrations.
 DESIGN In a double-blind, 6-wk study, 55 postmenopausal women aged 47-72 y were randomly assigned to 1 of 4 soy protein (40 g/d) isolate treatments: native phytate and native isoflavone (n = 14), native phytate and low isoflavone (n = 13), low phytate and native isoflavone (n = 14), or low phytate and low isoflavone (n = 14). We measured iron indexes, tHcy, CRP, and BMI.
 RESULTS Soy protein with native phytate significantly reduced tHcy (P = 0.017), transferrin saturation (P = 0.027), and ferritin (P = 0.029), whereas soy protein with native isoflavones had no effect on any variables. At baseline, BMI was highly correlated with tHcy (r = 0.39, P = 0.003) and CRP (r = 0.55, P < 0.0001), whereas HDL cholesterol was correlated with CRP (r = -0.30, P = 0.02). Multiple regression analysis showed that LDL cholesterol and BMI contributed significantly (R2= 19.9%, P = 0.003) to the overall variance in tHcy.
 CONCLUSION Consuming phytate-rich foods and maintaining a healthy weight may reduce atherosclerotic CVD risk factors in postmenopausal women.

title: Development and validation of QRISK3 risk prediction algorithms to estimate future risk of cardiovascular disease: prospective cohort study

text: Objectives To develop and validate updated QRISK3 prediction algorithms to estimate the 10 year risk of cardiovascular disease in women and men accounting for potential new risk factors. Design Prospective open cohort study. Setting General practices in England providing data for the QResearch database. Participants 1309 QResearch general practices in England: 981 practices were used to develop the scores and a separate set of 328 practices were used to validate the scores. 7.89 million patients aged 25-84 years were in the derivation cohort and 2.67 million patients in the validation cohort. Patients were free of cardiovascular disease and not prescribed statins at baseline. Methods Cox proportional hazards models in the derivation cohort to derive separate risk equations in men and women for evaluation at 10 years. Risk factors considered included those already in QRISK2 (age, ethnicity, deprivation, systolic blood pressure, body mass index, total cholesterol: high density lipoprotein cholesterol ratio, smoking, family history of coronary heart disease in a first degree relative aged less than 60 years, type 1 diabetes, type 2 diabetes, treated hypertension, rheumatoid arthritis, atrial fibrillation, chronic kidney disease (stage 4 or 5)) and new risk factors (chronic kidney disease (stage 3, 4, or 5), a measure of systolic blood pressure variability (standard deviation of repeated measures), migraine, corticosteroids, systemic lupus erythematosus (SLE), atypical antipsychotics, severe mental illness, and HIV/AIDs). We also considered erectile dysfunction diagnosis or treatment in men. Measures of calibration and discrimination were determined in the validation cohort for men and women separately and for individual subgroups by age group, ethnicity, and baseline disease status. Main outcome measures Incident cardiovascular disease recorded on any of the following three linked data sources: general practice, mortality, or hospital admission records. Results 363 565 incident cases of cardiovascular disease were identified in the derivation cohort during follow-up arising from 50.8 million person years of observation. All new risk factors considered met the model inclusion criteria except for HIV/AIDS, which was not statistically significant. The models had good calibration and high levels of explained variation and discrimination. In women, the algorithm explained 59.6% of the variation in time to diagnosis of cardiovascular disease (R2, with higher values indicating more variation), and the D statistic was 2.48 and Harrell's C statistic was 0.88 (both measures of discrimination, with higher values indicating better discrimination). The corresponding values for men were 54.8%, 2.26, and 0.86. Overall performance of the updated QRISK3 algorithms was similar to the QRISK2 algorithms. Conclusion Updated QRISK3 risk prediction models were developed and validated. The inclusion of additional clinical variables in QRISK3 (chronic kidney disease, a measure of systolic blood pressure variability (standard deviation of repeated measures), migraine, corticosteroids, SLE, atypical antipsychotics, severe mental illness, and erectile dysfunction) can help enable doctors to identify those at most risk of heart disease and stroke.

title: Vegan proteins may reduce risk of cancer, obesity, and cardiovascular disease by promoting increased glucagon activity.

text: Amino acids modulate the secretion of both insulin and glucagon; the composition of dietary protein therefore has the potential to influence the balance of glucagon and insulin activity. Soy protein, as well as many other vegan proteins, are higher in non-essential amino acids than most animal-derived food proteins, and as a result should preferentially favor glucagon production. Acting on hepatocytes, glucagon promotes (and insulin inhibits) cAMP-dependent mechanisms that down-regulate lipogenic enzymes and cholesterol synthesis, while up-regulating hepatic LDL receptors and production of the IGF-I antagonist IGFBP-1. The insulin-sensitizing properties of many vegan diets--high in fiber, low in saturated fat--should amplify these effects by down-regulating insulin secretion. Additionally, the relatively low essential amino acid content of some vegan diets may decrease hepatic IGF-I synthesis. Thus, diets featuring vegan proteins can be expected to lower elevated serum lipid levels, promote weight loss, and decrease circulating IGF-I activity. The latter effect should impede cancer induction (as is seen in animal studies with soy protein), lessen neutrophil-mediated inflammatory damage, and slow growth and maturation in children. In fact, vegans tend to have low serum lipids, lean physiques, shorter stature, later puberty, and decreased risk for certain prominent 'Western' cancers; a vegan diet has documented clinical efficacy in rheumatoid arthritis. Low-fat vegan diets may be especially protective in regard to cancers linked to insulin resistance--namely, breast and colon cancer--as well as prostate cancer; conversely, the high IGF-I activity associated with heavy ingestion of animal products may be largely responsible for the epidemic of 'Western' cancers in wealthy societies. Increased phytochemical intake is also likely to contribute to the reduction of cancer risk in vegans. Regression of coronary stenoses has been documented during low-fat vegan diets coupled with exercise training; such regimens also tend to markedly improve diabetic control and lower elevated blood pressure. Risk of many other degenerative disorders may be decreased in vegans, although reduced growth factor activity may be responsible for an increased risk of hemorrhagic stroke. By altering the glucagon/insulin balance, it is conceivable that supplemental intakes of key non-essential amino acids could enable omnivores to enjoy some of the health advantages of a vegan diet. An unnecessarily high intake of essential amino acids--either in the absolute sense or relative to total dietary protein--may prove to be as grave a risk factor for 'Western' degenerative diseases as is excessive fat intake.

title: Vegetarian vs. conventional diabetic diet – A 1-year follow-up

text: Abstract Objective Our previous 6-month, randomized study demonstrated the beneficial effect of a vegetarian (V) compared to a conventional diet (C) with similar caloric restriction on cardiovascular risk factors for patients with type 2 diabetes (T2D), namely increased insulin sensitivity, reduced body weight, reduced volume of visceral and subcutaneous fat, decreased LDL-cholesterol and improved oxidative stress markers and chosen adipokines. We conducted post-trial monitoring to determine whether the improved outcomes persisted 1 year after the end of the study. Methods 62 subjects with T2D who completed the study were asked to come for a 1-year follow-up to measure weight, waist circumference, HbA1c and blood lipids. No attempts were made to maintain their previously assigned diets. Results 44 patients (71%) attended the post-trial monitoring. Hypoglycemic agents were increased by 14% in V and by 26% in C; insulin therapy was introduced in 5% in V and in 13% in C one year after the end of the intervention. Neither weight nor waist circumference changed significantly in either group. HbA1c increased ( p  ≤ 0.05) similarly in both groups (+0.49 ± 1.04% in V vs. +0.42 ± 0.8% in C). Blood lipids did not change in either group. Conclusion One year after the end of the intervention, the positive effects of a vegetarian diet on cardiovascular risk factors compared to a conventional diet were partially maintained.

title: Cold Exposure Promotes Atherosclerotic Plaque Growth and Instability via UCP1-Dependent Lipolysis

text: Molecular mechanisms underlying the cold-associated high cardiovascular risk remain unknown. Here, we show that the cold-triggered food-intake-independent lipolysis significantly increased plasma levels of small low-density lipoprotein (LDL) remnants, leading to accelerated development of atherosclerotic lesions in mice. In two genetic mouse knockout models (apolipoprotein E(-/-) [ApoE(-/-)] and LDL receptor(-/-) [Ldlr(-/-)] mice), persistent cold exposure stimulated atherosclerotic plaque growth by increasing lipid deposition. Furthermore, marked increase of inflammatory cells and plaque-associated microvessels were detected in the cold-acclimated ApoE(-/-) and Ldlr(-/-) mice, leading to plaque instability. Deletion of uncoupling protein 1 (UCP1), a key mitochondrial protein involved in thermogenesis in brown adipose tissue (BAT), in the ApoE(-/-) strain completely protected mice from the cold-induced atherosclerotic lesions. Cold acclimation markedly reduced plasma levels of adiponectin, and systemic delivery of adiponectin protected ApoE(-/-) mice from plaque development. These findings provide mechanistic insights on low-temperature-associated cardiovascular risks.


query: LRBA prevents CTLA - 4 recycling.

title: Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations

text: The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

title: High CTLA-4 expression on Th17 cells results in increased sensitivity to CTLA-4 coinhibition and resistance to belatacept.

text: The CD28/cytotoxic T-lymphocyte antigen 4 (CTLA-4)blocker belatacept selectively inhibits alloreactive T cell responses but is associated with a high incidence of acute rejection following renal transplantation,which led us to investigate the etiology of belatacept–resistant graft rejection. T cells can differentiate into functionally distinct subsets of memory T cellsthat collectively enable protection against diverse classes of pathogens and can cross-react with allogeneicantigen and mediate graft rejection. T helper 17(Th17) cells are a pro-inflammatory CD4+ lineage that provides immunity to pathogens and are pathogenic in autoimmune disease. We found that T helper 1 (Th1)and Th17 memory compartments contained a similar frequency of divided cells following allogeneic stimulation. Compared to Th1 cells, Th17 memory cells expressed significantly higher levels of the coinhibitory molecule CTLA-4. Stimulation in the presence of belatacept inhibited Th1 responses but augmented Th17 cells due to greater sensitivity to coinhibition by CTLA-4. Th17 cells from renal transplant recipients were resistant to ex vivo CD28/CTLA-4 blockade with belatacept, and an elevated frequency of Th17 memory cells was associated with acute rejection during belatacept therapy. These data highlight important differences in costimulatory and coinhibitory requirements of CD4+ memory subsets, and demonstrate that the heterogeneity of pathogen-derived memory has implications for immunomodulation strategies.

title: CD28 and ITK signals regulate autoreactive T cell trafficking

text: Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

title: Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs.

text: Histone/protein deacetylases (HDACs) decrease histone and protein acetylation, typically leading to suppression of gene transcription and modulation of various protein functions. We found significant differences in expression of HDAC before and after stimulation of human T regulatory (Treg) and T effector cells, suggesting the potential for future selective targeting of Tregs with HDAC inhibitors (HDACi). Use of various HDACi small molecules enhanced, by up to 4.5-fold (average 2-fold), the suppressive functions of both freshly isolated and expanded human Tregs, consistent with our previous murine data. HDACi use increased Treg expression of CTLA-4, a key negative regulator of immune response, and we found a direct and significant correlation between CTLA-4 expression and Treg suppression. Hence, HDACi compounds are promising pharmacologic tools to increase Treg suppressive functions, and this action may potentially be of use in patients with autoimmunity or post-transplantation.

title: A Screen for Endocytic Motifs

text: Sorting signals for cargo selection into coated vesicles are usually in the form of short linear motifs. Three motifs for clathrin-mediated endocytosis have been identified: YXXPhi, [D/E]XXXL[L/I] and FXNPXY. To search for new endocytic motifs, we made a library of CD8 chimeras with random sequences in their cytoplasmic tails, and used a novel fluorescence-activated cell sorting (FACS)-based assay to select for endocytosed constructs. Out of the five tails that were most efficiently internalized, only one was found to contain a conventional motif. Two contain dileucine-like sequences that appear to be variations on the [D/E]XXXL[L/I] motif. Another contains a novel internalization signal, YXXXPhiN, which is able to function in cells expressing a mutant mu2 that cannot bind YXXPhi, indicating that it is not a variation on the YXXPhi motif. Similar sequences are present in endogenous proteins, including a functional YXXXPhiN (in addition to a classical YXXPhi) in cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Thus, the repertoire of endocytic motifs is more extensive than the three well-characterized sorting signals.

title: AP-1/sigma1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory.

text: Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1-sigma1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1-sigma1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three sigma1 subunit isoforms: A, B and C. The expressions of sigma1A and sigma1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from sigma1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The sigma1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation.

title: CD4+ T cell-mediated cytotoxicity eliminates primary tumor cells in metastatic melanoma through high MHC class II expression and can be enhanced by inhibitory receptor blockade

text: Metastatic melanoma is a rapidly progressing disease with high mortality rate and limited treatment options. Immunotherapy based on tumor-targeting cytotoxic T cell responses represents a promising strategy. To assist in its development, we examined the possibility and efficacy of using CD4+ cytotoxic T cells. The regulatory mechanisms controlling CD4+ T cell-mediated cytotoxicity were also investigated. We found that naturally occurring granzyme B and perforin-expressing CD4+ cytotoxic T cells can be recovered from metastatic melanoma patients at significantly elevated frequencies compared to those from healthy controls. These CD4+ cytotoxic T cells were also capable of killing autologous tumor cells harvested from metastatic melanoma, independent of CD8+ T cells or any other cell types. However, several restricting factors were observed. First, the cytolytic activity by CD4+ T cells required high MHC class II expression on melanoma cells, which was not satisfied in a subset of melanomas. Second, the granzyme B and perforin release by activated CD4+ cytotoxic T cells was reduced after coculturing with autologous melanoma cells, characterized by low LAMP-1 expression and low granzyme B and perforin secretion in the supernatant. This suggested that inhibitory mechanisms were present to suppress CD4+ cytotoxic T cells. Indeed, blockade of PD-1 and CTLA-4 had increased the cytolytic activity of CD4+ T cells but was only effective in MHC class II high but not MHC class II low melanomas. Together, our study showed that CD4+ T cell-mediated cytotoxicity could eliminate primary melanoma cells but the efficacy depended on MHC class II expression.

title: Effect of interleukin-1 and tumor necrosis factor/cachectin on glucose turnover in the rat.

text: We studied the effect of recombinant human interleukin-1 beta (IL-1) and recombinant human tumor necrosis factor alpha/cachectin (TNF) on glucose kinetics in healthy rats by means of a primed constant infusion of D-(6-3H)glucose and D-[U-14C]glucose. During the isotope (6-hour) and monokine (4-hour) infusion, plasma levels of glucagon and insulin were determined and correlated with changes in glucose metabolism. The rates of glucose appearance (Ra) and disappearance (Rd) were elevated only with IL-1 and were associated with an increase in glucagon and a concomitant decrease in the ratio of insulin to glucagon. Plasma glucose concentration was increased early after IL-1 administration and coincided with the peak in the Ra. The augmentation of the metabolic clearance rate (MCR) and percent of flux oxidized by IL-1 suggest that this monokine induces the utilization of glucose as a substrate. TNF administration failed to modify the Ra or Rd, percent of flux oxidized, or MCR. TNF-treated rats increased the percent of glucose recycling, but not the total rate of glucose production. The results of this experiment suggest that endogenous macrophage products participate in the diverse alterations of carbohydrate metabolism seen during injury and/or infection.

title: Updated energy budgets for neural computation in the neocortex and cerebellum.

text: The brain's energy supply determines its information processing power, and generates functional imaging signals. The energy use on the different subcellular processes underlying neural information processing has been estimated previously for the grey matter of the cerebral and cerebellar cortex. However, these estimates need reevaluating following recent work demonstrating that action potentials in mammalian neurons are much more energy efficient than was previously thought. Using this new knowledge, this paper provides revised estimates for the energy expenditure on neural computation in a simple model for the cerebral cortex and a detailed model of the cerebellar cortex. In cerebral cortex, most signaling energy (50%) is used on postsynaptic glutamate receptors, 21% is used on action potentials, 20% on resting potentials, 5% on presynaptic transmitter release, and 4% on transmitter recycling. In the cerebellar cortex, excitatory neurons use 75% and inhibitory neurons 25% of the signaling energy, and most energy is used on information processing by non-principal neurons: Purkinje cells use only 15% of the signaling energy. The majority of cerebellar signaling energy use is on the maintenance of resting potentials (54%) and postsynaptic receptors (22%), while action potentials account for only 17% of the signaling energy use.

title: Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions

text: Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.

title: The dystonia-associated protein torsinA modulates synaptic vesicle recycling.

text: The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.

title: How nascent phagosomes mature to become phagolysosomes.

text: Phagocytosis mediates the clearance of apoptotic bodies and also the elimination of microbial pathogens. The nascent phagocytic vacuole formed upon particle engulfment lacks microbicidal and degradative activity. These capabilities are acquired as the phagosome undergoes maturation; a progressive remodeling of its membrane and contents that culminates in the formation of phagolysosomes. Maturation entails orderly sequential fusion of the phagosomal vacuole with specialized endocytic and secretory compartments. Concomitantly, the phagosomal membrane undergoes both inward and outward vesiculation and tubulation followed by fission, thereby recycling components and maintaining its overall size. Here, we summarize what is known about the molecular machinery that governs this complex metamorphosis of phagosome maturation.

title: Incidence of adverse drug events and potential adverse drug events. Implications for prevention. ADE Prevention Study Group.

text:
OBJECTIVES To assess incidence and preventability of adverse drug events (ADEs) and potential ADEs. To analyze preventable events to develop prevention strategies.
 DESIGN Prospective cohort study.
 PARTICIPANTS All 4031 adult admissions to a stratified random sample of 11 medical and surgical units in two tertiary care hospitals over a 6-month period. Units included two medical and three surgical intensive care units and four medical and two surgical general care units.
 MAIN OUTCOME MEASURES Adverse drug events and potential ADEs.
 METHODS Incidents were detected by stimulated self-report by nurses and pharmacists and by daily review of all charts by nurse investigators. Incidents were subsequently classified by two independent reviewers as to whether they represented ADEs or potential ADEs and as to severity and preventability.
 RESULTS Over 6 months, 247 ADEs and 194 potential ADEs were identified. Extrapolated event rates were 6.5 ADEs and 5.5 potential ADEs per 100 nonobstetrical admissions, for mean numbers per hospital per year of approximately 1900 ADEs and 1600 potential ADEs. Of all ADEs, 1% were fatal (none preventable), 12% life-threatening, 30% serious, and 57% significant. Twenty-eight percent were judged preventable. Of the life-threatening and serious ADEs, 42% were preventable, compared with 18% of significant ADEs. Errors resulting in preventable ADEs occurred most often at the stages of ordering (56%) and administration (34%); transcription (6%) and dispensing errors (4%) were less common. Errors were much more likely to be intercepted if the error occurred earlier in the process: 48% at the ordering stage vs 0% at the administration stage.
 CONCLUSION Adverse drug events were common and often preventable; serious ADEs were more likely to be preventable. Most resulted from errors at the ordering stage, but many also occurred at the administration stage. Prevention strategies should target both stages of the drug delivery process.

title: Increased CDK1 activity determines the timing of kinetochore-microtubule attachments in meiosis I

text: Chromosome segregation during cell division depends on stable attachment of kinetochores to spindle microtubules. Mitotic spindle formation and kinetochore-microtubule (K-MT) capture typically occur within minutes of nuclear envelope breakdown. In contrast, during meiosis I in mouse oocytes, formation of the acentrosomal bipolar spindle takes 3-4 h, and stabilization of K-MT attachments is delayed an additional 3-4 h. The mechanism responsible for this delay, which likely prevents stabilization of erroneous attachments during spindle formation, is unknown. Here we show that during meiosis I, attachments are regulated by CDK1 activity, which gradually increases through prometaphase and metaphase I. Partial reduction of CDK1 activity delayed formation of stable attachments, whereas a premature increase in CDK1 activity led to precocious formation of stable attachments and eventually lagging chromosomes at anaphase I. These results indicate that the slow increase in CDK1 activity in meiosis I acts as a timing mechanism to allow stable K-MT attachments only after bipolar spindle formation, thus preventing attachment errors.

title: Dexamethasone and risk of nausea and vomiting and postoperative bleeding after tonsillectomy in children: a randomized trial.

text:
CONTEXT Dexamethasone is widely used to prevent postoperative nausea and vomiting (PONV) in pediatric tonsillectomy.
 OBJECTIVE To assess whether dexamethasone dose-dependently reduces the risk of PONV at 24 hours after tonsillectomy.
 DESIGN, SETTING, AND PATIENTS Randomized placebo-controlled trial conducted among 215 children undergoing elective tonsillectomy at a major public teaching hospital in Switzerland from February 2005 to December 2007.
 INTERVENTIONS Children were randomly assigned to receive dexamethasone (0.05, 0.15, or 0.5 mg/kg) or placebo intravenously after induction of anesthesia. Acetaminophen-codeine and ibuprofen were given as postoperative analgesia. Follow-up continued until the 10th postoperative day.
 MAIN OUTCOME MEASURES The primary end point was prevention of PONV at 24 hours; secondary end points were decrease in the need for ibuprofen at 24 hours and evaluation of adverse effects.
 RESULTS At 24 hours, 24 of 54 participants who received placebo (44%; 95% confidence interval [CI], 31%-59%) had experienced PONV compared with 20 of 53 (38%; 95% CI, 25%-52%), 13 of 54 (24%; 95% CI, 13%-38%), and 6 of 52 (12%; 95% CI, 4%-23%) who received dexamethasone at 0.05, 0.15, and 0.5 mg/kg, respectively (P<.001 for linear trend). Children who received dexamethasone received significantly less ibuprofen. There were 26 postoperative bleeding episodes in 22 children. Two of 53 (4%; 95% CI, 0.5%-13%) children who received placebo had bleeding compared with 6 of 53 (11%; 95% CI, 4%-23%), 2 of 51 (4%; 95% CI, 0.5%-13%), and 12 of 50 (24%; 95% CI, 13%-38%) who received dexamethasone at 0.05, 0.15, and 0.5 mg/kg, respectively (P = .003). Dexamethasone, 0.5 mg/kg, was associated with the highest bleeding risk (adjusted relative risk, 6.80; 95% CI, 1.77-16.5). Eight children had to undergo emergency reoperation because of bleeding, all of whom had received dexamethasone. The trial was stopped early for safety reasons.
 CONCLUSION In this study of children undergoing tonsillectomy, dexamethasone decreased the risk of PONV dose dependently but was associated with an increased risk of postoperative bleeding.
 TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00403806.

title: Endocytic Trafficking of Rac Is Required for the Spatial Restriction of Signaling in Cell Migration

text: The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.

title: A direct role for Met endocytosis in tumorigenesis

text: Compartmentalization of signals generated by receptor tyrosine kinase (RTK) endocytosis has emerged as a major determinant of various cell functions. Here, using tumour-associated Met-activating mutations, we demonstrate a direct link between endocytosis and tumorigenicity. Met mutants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes, activation of the GTPase Rac1, loss of actin stress fibres and increased levels of cell migration. Blocking endocytosis inhibited mutants’ anchorage-independent growth, in vivo tumorigenesis and metastasis while maintaining their activation. One mutant resistant to inhibition by a Met-specific tyrosine kinase inhibitor was sensitive to endocytosis inhibition. Thus, oncogenicity of Met mutants results not only from activation but also from their altered endocytic trafficking, indicating that endosomal signalling may be a crucial mechanism regulating RTK-dependent tumorigenesis.

title: A view to a kill: the bacterial type VI secretion system.

text: The bacterial type VI secretion system (T6SS) is an organelle that is structurally and mechanistically analogous to an intracellular membrane-attached contractile phage tail. Recent studies determined that a rapid conformational change in the structure of a sheath protein complex propels T6SS spike and tube components along with antibacterial and antieukaryotic effectors out of predatory T6SS(+) cells and into prey cells. The contracted organelle is then recycled in an ATP-dependent process. T6SS is regulated at transcriptional and posttranslational levels, the latter involving detection of membrane perturbation in some species. In addition to directly targeting eukaryotic cells, the T6SS can also target other bacteria coinfecting a mammalian host, highlighting the importance of the T6SS not only for bacterial survival in environmental ecosystems, but also in the context of infection and disease. This review highlights these and other advances in our understanding of the structure, mechanical function, assembly, and regulation of the T6SS.

title: Autophagy and its potent modulators from phytochemicals in cancer treatment

text: Autophagy is a ubiquitous catabolic process by which damaged or harmful intracellular components are delivered to the lysosomes for self-digestion and recycling. It is critical in cancer treatment. Therapy-induced autophagy predominantly acts as a pro-survival mechanism, but progressive autophagy can lead to non-apoptotic cell death, also known as autophagic cell death. Plants or herbs contain various natural compounds that are widely used in the treatment of many types of malignancies. Emerging evidence indicates that phytochemicals targeting the autophagic pathway are promising agents for cancer treatment. However, these compounds play different roles in autophagy. In this review, we discussed the role of autophagy in cancer development and therapy, and focussed on elucidating the anti-cancer activities of autophagic modulators, especially phytochemicals. Notably, we described a novel premise that the dynamic role of phytochemicals should be evaluated in regulation of autophagy in cancer.

title: Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels.

text: To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead displacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response; (2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces; (3) later adaptive cell stiffening with sustained (>15 second) static stresses; and (4) a large-scale repositioning response with prolonged (>1 minute) stress. Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by chemically dissipating cytoskeletal prestress (isometric tension), whereas the later adaptive response was not. The repositioning response was prevented by inhibiting tension through interference with Rho signaling, similar to the case of the immediate and early responses, but it was also prevented by blocking mechanosensitive ion channels or by inhibiting Src tyrosine kinases. All adaptive responses were suppressed by cooling cells to 4 degrees C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.


query: LRBA promotes CTLA - 4 recycling.

title: Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations

text: The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

title: High CTLA-4 expression on Th17 cells results in increased sensitivity to CTLA-4 coinhibition and resistance to belatacept.

text: The CD28/cytotoxic T-lymphocyte antigen 4 (CTLA-4)blocker belatacept selectively inhibits alloreactive T cell responses but is associated with a high incidence of acute rejection following renal transplantation,which led us to investigate the etiology of belatacept–resistant graft rejection. T cells can differentiate into functionally distinct subsets of memory T cellsthat collectively enable protection against diverse classes of pathogens and can cross-react with allogeneicantigen and mediate graft rejection. T helper 17(Th17) cells are a pro-inflammatory CD4+ lineage that provides immunity to pathogens and are pathogenic in autoimmune disease. We found that T helper 1 (Th1)and Th17 memory compartments contained a similar frequency of divided cells following allogeneic stimulation. Compared to Th1 cells, Th17 memory cells expressed significantly higher levels of the coinhibitory molecule CTLA-4. Stimulation in the presence of belatacept inhibited Th1 responses but augmented Th17 cells due to greater sensitivity to coinhibition by CTLA-4. Th17 cells from renal transplant recipients were resistant to ex vivo CD28/CTLA-4 blockade with belatacept, and an elevated frequency of Th17 memory cells was associated with acute rejection during belatacept therapy. These data highlight important differences in costimulatory and coinhibitory requirements of CD4+ memory subsets, and demonstrate that the heterogeneity of pathogen-derived memory has implications for immunomodulation strategies.

text: Histone/protein deacetylases (HDACs) decrease histone and protein acetylation, typically leading to suppression of gene transcription and modulation of various protein functions. We found significant differences in expression of HDAC before and after stimulation of human T regulatory (Treg) and T effector cells, suggesting the potential for future selective targeting of Tregs with HDAC inhibitors (HDACi). Use of various HDACi small molecules enhanced, by up to 4.5-fold (average 2-fold), the suppressive functions of both freshly isolated and expanded human Tregs, consistent with our previous murine data. HDACi use increased Treg expression of CTLA-4, a key negative regulator of immune response, and we found a direct and significant correlation between CTLA-4 expression and Treg suppression. Hence, HDACi compounds are promising pharmacologic tools to increase Treg suppressive functions, and this action may potentially be of use in patients with autoimmunity or post-transplantation.

title: A Screen for Endocytic Motifs

title: CD28 and ITK signals regulate autoreactive T cell trafficking

text: Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

title: Kinetic Analysis Reveals the Fate of a MicroRNA following Target Regulation in Mammalian Cells

text: Considerable details about microRNA (miRNA) biogenesis and regulation have been uncovered, but little is known about the fate of the miRNA subsequent to target regulation. To gain insight into this process, we carried out kinetic analysis of a miRNA's turnover following termination of its biogenesis and during regulation of a target that is not subject to Ago2-mediated catalytic cleavage. By quantitating the number of molecules of the miRNA and its target in steady state and in the course of its decay, we found that each miRNA molecule was able to regulate at least two target transcripts, providing in vivo evidence that the miRNA is not irreversibly sequestered with its target and that the nonslicing pathway of miRNA regulation is multiple-turnover. Using deep sequencing, we further show that miRNA recycling is limited by target regulation, which promotes posttranscriptional modifications to the 3' end of the miRNA and accelerates the miRNA's rate of decay. These studies provide new insight into the efficiency of miRNA regulation that help to explain how a miRNA can regulate a vast number of transcripts and that identify one of the mechanisms that impart specificity to miRNA decay in mammalian cells.

title: Chemokine-like receptor 1 (CMKLR1) and chemokine (C-C motif) receptor-like 2 (CCRL2); two multifunctional receptors with unusual properties.

text: Chemokine-like receptor 1 (CMKLR1), also known as ChemR23, and chemokine (C-C motif) receptor-like 2 (CCRL2) are 7-transmembrane receptors that were cloned in the late 1990s based on their homology to known G-protein-coupled receptors. They were previously orphan receptors without any known biological roles; however, recent studies identified ligands for these receptors and their functions have begun to be unveiled. The plasma protein-derived chemoattractant chemerin is a ligand for CMKLR1 and activation of CMKLR1 with chemerin induces the migration of macrophages and dendritic cells (DCs) in vitro, suggesting a proinflammatory role. However, in vivo studies using CMKLR-deficient mice suggest an anti-inflammatory role for this receptor, possibly due to the recruitment of tolerogenic plasmacytoid DCs. Chemerin/CMKLR1 interaction also promotes adipogenesis and angiogenesis. The anti-inflammatory lipid mediator, resolving E1, is another CMKLR1 ligand and it inhibits leukocyte infiltration and proinflammatory gene expression. These divergent results suggest that CMKLR1 is a multifunctional receptor. The chemokine CCL5 and CCL19 are reported to bind to CCRL2. Like Duffy antigen for chemokine receptor (DARC), D6 and CCX-CKR, CCRL2 does not signal, but it constitutively recycles, potentially reducing local concentration of CCL5 and CCL19 and subsequent immune responses. Surprisingly, chemerin, a ligand for CMKLR1, is a ligand for CCRL2. CCRL2 binds chemerin and increases local chemerin concentration to efficiently present it to CMKLR1 on nearby cells, providing a link between CCRL2 and CMKLR1. Although these findings suggest an anti-inflammatory role, a recent study using CCRL2-deficient mice indicates a proinflammatory role; thus, CCRL2 may also be multifunctional. Further studies using CMKLR1- or CCRL2-deficient mice are needed to further define the role of these receptors in immune responses and other cellular processes.

title: The Proto-Oncogene c-maf Is Responsible for Tissue-Specific Expression of Interleukin-4

text: The molecular basis for the distinctive cytokine expression of CD4+ T helper 1 (Th1) and T helper 2 (Th2) subsets remains elusive. Here, we report that the proto-oncogene c-maf, a basic region/leucine zipper transcription factor, controls tissue-specific expression of IL-4. c-Maf is expressed in Th2 but not Th1 clones and is induced during normal precursor cell differentiation along a Th2 but not Th1 lineage. c-Maf binds to a c-Maf response element (MARE) in the proximal IL-4 promoter adjacent to a site footprinted by extracts from Th2 but not Th1 clones. Ectopic expression of c-Maf transactivates the IL-4 promoter in Th1 cells, B cells, and nonlymphoid cells, a function that maps to the MARE and Th2-specific footprint. Furthermore, c-Maf acts in synergy with the nuclear factor of activated T cells (NF-ATp) to initiate endogeneous IL-4 production by B cells. Manipulation of c-Maf may alter Th subset ratios in human disease.

title: GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.

text: Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.