Tracking biochemical changes induced by iron loading in AML12 cells with synchrotron live cell, time-lapse infrared microscopy.

Hepatocytes are essential for maintaining the homeostasis of iron and lipid metabolism in mammals. Dysregulation of either iron or lipids has been linked with serious health consequences, including non-alcoholic fatty liver disease (NAFLD). Considered the hepatic manifestation of metabolic syndrome, NAFLD is characterised by dysregulated lipid metabolism leading to a lipid storage phenotype. Mild to moderate increases in hepatic iron have been observed in ∼30% of individuals with NAFLD; however, direct observation of the mechanism behind this increase has remained elusive. To address this issue, we sought to determine the metabolic consequences of iron loading on cellular metabolism using live cell, time-lapse Fourier transform infrared (FTIR) microscopy utilising a synchrotron radiation source to track biochemical changes. The use of synchrotron FTIR is non-destructive and label-free, and allowed observation of spatially resolved, sub-cellular biochemical changes over a period of 8 h. Using this approach, we have demonstrated that iron loading in AML12 cells induced perturbation of lipid metabolism congruent with steatosis development. Iron-loaded cells had approximately three times higher relative ester carbonyl concentration compared with controls, indicating an accumulation of triglycerides. The methylene/methyl ratio qualitatively suggests the acyl chain length of fatty acids in iron-loaded cells increased over the 8 h period of monitoring compared with a reduction observed in the control cells. Our findings provide direct evidence that mild to moderate iron loading in hepatocytes drives de novo lipid synthesis, consistent with a role for iron in the initial hepatic lipid accumulation that leads to the development of hepatic steatosis.

Butyrate generated by gut microbiota and its therapeutic role in metabolic syndrome.

Metabolic syndrome (MetS) and the associated incidence of cardiovascular disease and type 2 diabetes represents a significant contributor to morbidity and mortality worldwide. Butyrate, a short-chain fatty acid produced by the gut microbiome, has long been known to promote growth in farmed animals and more recently has been reported to improve body weight and composition, lipid profile, insulin sensitivity and glycaemia in animal models of MetS. In vitro studies have examined the influence of butyrate on intestinal cells, adipose tissue, skeletal muscle, hepatocytes, pancreatic islets and blood vessels, highlighting genes and pathways that may contribute to its beneficial effects. Butyrate's influences in these cells have been attributed primarily to its epigenetic effects as a histone deacetylase inhibitor, as well as its role as an agonist of free fatty acid receptors, but clear mechanistic evidence is lacking. There is also uncertainty whether results from animal studies can translate to human trials due to butyrate's poor systemic availability and rapid clearance. Hitherto, several small-scale human clinical trials have failed to show significant benefits in MetS patients. Further trials are clearly needed, including with formulations designed to improve butyrate's availability. Regardless, dietary intervention to increase the rate of butyrate production may be a beneficial addition to current treatment. This review outlines the current body of evidence on the suitability of butyrate supplementation for MetS, looking at mechanistic effects on the various components of MetS and highlighting gaps in the knowledge and roadblocks to its use in humans.

Pleiotropic and Adverse Effects of Statins-Do Epigenetics Play a Role?

Statins are widely used to prevent major cardiovascular events by lowering serum cholesterol. There is evidence that statins have pleiotropic effects-that is, cholesterol-independent effects-that may also confer protection from cardiovascular disease and potentially numerous other pathologies, including cancer. Statins also have a number of well described adverse effects, including myopathy, rhabdomyolysis, liver damage, and type 2 diabetes. This paper examines the evidence of epigenetic modifications as a contributory factor to the pleiotropic and adverse effects of statins. In vitro and animal studies have shown that statins can inhibit histone deacetylase activity and increase histone acetylation. Similarly, there is evidence that statins may inhibit both histone and DNA methyltransferases and subsequently demethylate histone residues and DNA, respectively. These changes have been shown to alter expression of various genes, including tumor suppressor genes and genes thought to have anti-atherosclerotic actions. Statins have also been shown to influence the expression of numerous microRNAs that suppress the translation of proteins involved in tumorigenesis and vascular function. Whether the adverse effects of statins may also have an epigenetic component has been less widely studied, although there is evidence that microRNA expression may be altered in statin-induced muscle and liver damage. As epigenetics and microRNAs influence gene expression, these changes could contribute to the pleiotropic and adverse effects of statins and have long-lasting effects on the health of statin users.

Nucleic acid reference materials (NARMs): definitions and issues.

Molecular diagnostics is one of the most rapidly growing areas of laboratory medicine. This rapid growth of clinical molecular tests has outpaced the availability and development of reference methods and reference materials. Such methods and materials are important for the development, validation, and interpretation of diagnostic methods and tests. Yet, there is a lack of harmonization between the numerous international organizations currently either certifying or defining reference materials. The objective of this position paper is to review and clarify the definition, attributes and applications for the use of reference materials in the context of molecular diagnostics.

Atorvastatin increases expression of low-density lipoprotein receptor mRNA in human circulating mononuclear cells.

1. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors, or statins, are commonly used to lower plasma cholesterol levels. HMGCR and the low-density lipoprotein (LDL) receptor (LDLR) are of central importance to cholesterol homeostasis and yet there is a paucity of data on the effect of statins on the regulation of the LDLR and HMGCR in humans. 2. In the present study, we examined the effect of atorvastatin on the expression of HMGCR, LDLR and LDLR-related protein (LRP) mRNA in circulating mononuclear cells. Twelve human volunteers were treated with atorvastatin, 20 mg/day for 4 weeks. 3. Atorvastatin decreased plasma total and LDL-cholesterol by 29% (P < 0.0001) and 41% (P < 0.001), respectively, and increased LDLR mRNA abundance, in absolute terms, by 35% (P < 0.001) and 31% (P < 0.0001) and 37% (P = 0.01) relative to reference GAPDH and beta-actin mRNA, respectively. In contrast, atorvastatin had no significant effect on LRP or HMGCR mRNA levels. 4. The increase in LDLR mRNA in circulating mononuclear cells agrees with the few human studies conducted, as well as with in vitro and animal studies, whereas the unchanged HMGCR mRNA is consistent with the hepatic specificity of atorvastatin. The present study firmly documents an increase in LDLR mRNA levels in response to statin administration in normal humans.

Statins Do Not Directly Inhibit the Activity of Major Epigenetic Modifying Enzymes.

The potential anticancer effects of statins-a widely used class of cholesterol lowering drugs-has generated significant interest, as has the use of epigenetic modifying drugs such as HDAC and DNMT inhibitors. We set out to investigate the effect of statin drugs on epigenetic modifications in multiple cell lines, including hepatocellular carcinoma, breast carcinoma, leukemic macrophages, cervical adenocarcinoma, and insulin-secreting cells, as well as liver extracts from statin-treated C57B1/6J mice. Cells or cell extracts were treated with statins and with established epigenetic modulators, and HDAC, HAT, and DNMT activities were quantified. We also examined histone acetylation by immunoblotting. Statins altered neither HDAC nor HAT activity. Accordingly, acetylation of histones H3 and H4 was unchanged with statin treatment. However, statins tended to increase DNMT activity. These results indicate that direct inhibition of the major classes of epigenetic modifying enzymes, as previously reported elsewhere, is unlikely to contribute to any anticancer effects of statins. This study concerned global effects on epigenetic enzyme activities and histone acetylation; whether statins influence epigenetic modifications in certain genomic regions, cannot be ruled out and remains to be investigated.

The Regulatory Status Adopted by Lymph Node Dendritic Cells and T Cells During Healthy Aging Is Maintained During Cancer and May Contribute to Reduced Responses to Immunotherapy.

Aging is associated with an increased incidence of cancer. One contributing factor could be modulation of immune cells responsible for anti-tumor responses, such as dendritic cells (DCs) and T cells. These immunological changes may also impact the efficacy of cancer immunotherapies in the elderly. The effects of healthy aging on DCs and T cells, and their impact on anti-mesothelioma immune responses, had not been reported. This study examined DCs and T cells in young (2-5 months; equivalent to 16-26 human years) and elderly (20-24 months; equivalent to 60-70 human years) healthy and mesothelioma-bearing C57BL/6J mice. During healthy aging, elderly lymph nodes adopted a regulatory profile, characterized by: (i) increased plasmacytoid DCs, (ii) increased expression of the adenosine-producing enzyme CD73 on CD11c+ cells, and (iii) increased expression of multiple regulatory markers (including CD73, the adenosine A2B receptor, CTLA-4, PD-1, ICOS, LAG-3, and IL-10) on CD8+ and CD4+ T cells, compared to lymph nodes from young mice. Although mesotheliomas grew faster in elderly mice, the increased regulatory status observed in healthy elderly lymph node DCs and T cells was not further exacerbated. However, elderly tumor-bearing mice demonstrated reduced MHC-I, MHC-II and CD80 on CD11c+ cells, and decreased IFN-γ by CD8+ and CD4+ T cells within tumors, compared to young counterparts, implying loss of function. An agonist CD40 antibody based immunotherapy was less efficient at promoting tumor regression in elderly mice, which may be due to: (i) failure of elderly CD8+ T cells to up-regulate perforin, and (ii) increased expression of multiple regulatory markers on CD11c+ cells and T cells in elderly tumor-draining lymph nodes (including CD73, PD-1, ICOS, LAG-3, and TGF-ß). Our findings suggest that checkpoint blockade may improve responses to immunotherapy in elderly hosts with mesothelioma, and warrants further investigation.

Elderly dendritic cells respond to LPS/IFN-γ and CD40L stimulation despite incomplete maturation.

There is evidence that dendritic cells (DCs) undergo age-related changes that modulate their function with their key role being priming antigen-specific effector T cells. This occurs once DCs develop into antigen-presenting cells in response to stimuli/danger signals. However, the effects of aging on DC responses to bacterial lipopolysaccharide (LPS), the pro-inflammatory cytokine interferon (IFN)-γ and CD40 ligand (CD40L) have not yet been systematically evaluated. We examined responses of blood myeloid (m)DC1s, mDC2s, plasmacytoid (p)DCs, and monocyte-derived DCs (MoDCs) from young (21-40 years) and elderly (60-84 years) healthy human volunteers to LPS/IFN-γ or CD40L stimulation. All elderly DC subsets demonstrated comparable up-regulation of co-stimulatory molecules (CD40, CD80 and/or CD86), intracellular pro-inflammatory cytokine levels (IFN-γ, tumour necrosis factor (TNF)-α, IL-6 and/or IL-12), and/or secreted cytokine levels (IFN-α, IFN-γ, TNF-α, and IL-12) to their younger counterparts. Furthermore, elderly-derived LPS/IFN-γ or CD40L-activated MoDCs induced similar or increased levels of CD8+ and CD4+ T cell proliferation, and similar T cell functional phenotypes, to their younger counterparts. However, elderly LPS/IFN-γ-activated MoDCs were unreliable in their ability to up-regulate chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and IL-6 secretion, implying an inability to dependably induce an inflammatory response. A key age-related difference was that, unlike young-derived MoDCs that completely lost their ability to process antigen, elderly-derived MoDCs maintained their antigen processing ability after LPS/IFN-γ maturation, measured using the DQ-ovalbumin assay; this response implies incomplete maturation that may enable elderly DCs to continuously present antigen. These differences may impact on the efficacy of anti-pathogen and anti-tumour immune responses in the elderly.

Modulation of dendritic cell and T cell cross-talk during aging: The potential role of checkpoint inhibitory molecules.

Dendritic cells (DCs) undergo continuous changes throughout life, and there is evidence that elderly DCs have a reduced capacity to stimulate T cells, which may contribute to impaired anti-tumour immune responses in elderly people with cancer. Changes in checkpoint inhibitory molecules/pathways during aging may be one mechanism that impairs the ability of elderly DCs to activate T cells. However, little is currently known regarding the combined effects of aging and cancer on DC and T cell inhibitory molecules/pathways. In this review, we discuss our current understanding of the influence of aging and cancer on key DC and T cell inhibitory molecules/pathways, the potential underlying cellular and molecular mechanisms contributing to their modulation, and the possibility of therapeutically targeting inhibitory molecules in elderly cancer patients.

Plasma cholesterol in adults with phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine (Phe) catabolism resulting from a deficiency of L-phenylalanine hydroxylase (PAH). An association between hyperphenylalaninaemia (HPA) and hypocholesterolaemia has been reported in children. However, controversy exists as to whether this is due to the low protein diet or to a disruption to cholesterol biosynthesis inherent to those with PKU. We investigated the relationship between blood Phe and plasma cholesterol in 41 apparently healthy adults with PKU (26 female, 15 male, age 18-57 years, median age 26 years) attending a PKU outpatient clinic at an adult tertiary care hospital. Of these patients, 33 (80%) were compliant with a Phe-restricted diet with amino acid supplementation, whereas eight (20%) were not. The PKU subjects had a mean body mass index (BMI) of 30.3 ±â€Š1.8 kg/m; 72% were obese, 14% overweight, with only 14% having normal BMI. The mean blood Phe was 1194 ±â€Š522 µmol/L with plasma total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol and apolipoprotein (apo) B concentrations of 4.3 ±â€Š0.8 mmol/L, 1.6 ±â€Š0.8 mmol/L, 1.2 ±â€Š0.3 mmol/L, 2.3 ±â€Š0.8 mmol/L, and 0.83 ±â€Š0.21 g/L, respectively. The mean LDL-cholesterol was 19% lower in PKU females than that of 8944 age-matched females from a community population (2.5 ±â€Š0.8 mmol/L vs. 3.1 ±â€Š0.9 mmol/L, p < 0.001). Similarly, the mean LDL-cholesterol was 32% lower in PKU males than 3786 age-matched males (2.1 ±â€Š0.7 mmol/L vs. 3.1 ±â€Š1.0 mmol/L, p < 0.0001). No correlations were observed between Phe and total cholesterol, LDL-cholesterol or apoB in the PKU cohort. Adults with PKU had low-normal cholesterol concentrations, with no correlation observed between Phe and cholesterol levels. Our findings support the concept that the HPA found in PKU, rather than an effect of a low-protein diet, leads to hypocholesterolaemia. Studies are required to determine whether this cholesterol-lowering effect confers cardioprotection.

Mesothelioma tumor cells modulate dendritic cell lipid content, phenotype and function.

Dendritic cells (DCs) play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs) were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay), upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4+ CD8α- DCs, CD4- CD8α- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8+ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.

Endothelial nitric oxide synthase gene polymorphism, homocysteine, cholesterol and vascular endothelial function.

Nitric oxide-dependent vasodilation is impaired early in the pathogenesis of vascular disease. Both the 4ab polymorphism of endothelial nitric oxide synthase (eNOS) and elevated plasma homocysteine are putatively associated with coronary artery disease (CAD). Few studies have investigated the influence of either on endothelial function in normal subjects. We aimed to examine any effect of three eNOS gene polymorphisms and plasma levels of homocysteine, folate and lipids on vascular endothelial function in normal healthy subjects. Community subjects (n=60) were selected for their eNOS genotype. Largely NOz.-dependent, flow-mediated dilation (FMD) of the brachial artery and the response to glyceryl trinitrate (GTN) were measured. Neither FMD nor response to GTN in 4a allele carriers was significantly different from that of 4b homozygotes, (7.1+/-0.5 S.E.M. vs. 7.1+/-0.6%) and (18.9+/-1.2 vs. 18.9+/-0.9%), respectively. Responses were also independent of the other polymorphisms. FMD was significantly correlated with HDL-cholesterol (P=0.02). After accounting for serum folate, there was a significant inverse correlation between FMD and plasma homocysteine (P=0.03). In these normal community subjects, plasma homocysteine and HDL-cholesterol were predictors of FMD despite subjects being recruited without regard to these variables and despite normal plasma levels.

Phenylketonuria: an inborn error of phenylalanine metabolism.

Phenylketonuria (PKU) is an autosomal recessive inborn error of phenylalanine (Phe) metabolism resulting from deficiency of phenylalanine hydroxylase (PAH). Most forms of PKU and hyperphenylalaninaemia (HPA) are caused by mutations in the PAH gene on chromosome 12q23.2. Untreated PKU is associated with an abnormal phenotype which includes growth failure, poor skin pigmentation, microcephaly, seizures, global developmental delay and severe intellectual impairment. However, since the introduction of newborn screening programs and with early dietary intervention, children born with PKU can now expect to lead relatively normal lives. A better understanding of the biochemistry, genetics and molecular basis of PKU, as well as the need for improved treatment options, has led to the development of new therapeutic strategies.

LDL-receptor mRNA expression in men is downregulated within an hour of an acute fat load and is influenced by genetic polymorphism.

Little is known about the immediate effects of dietary fat on the expression of genes involved in cholesterol metabolism in humans. We investigated the effects of a high-fat meal on circulating mononuclear cell messenger RNA (mRNA) for the LDL receptor (LDLR), LDLR-related protein (LRP), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) over 10 h. Selection of 12 C and 7 T homozygotes for the LRP exon 22 C200T polymorphism for the study also enabled us to examine the influence of this polymorphism on postprandial mRNA expression and lipoproteins, of relevance because of LRP's role in postprandial lipoprotein metabolism and association of the polymorphism with coronary artery disease. We found a postprandial decrease in LDLR mRNA abundance relative to the reference beta-actin (BA) mRNA. The decreased LDLR/BA mRNA value was apparent at 1 h (P < 0.005) and decreased to 25% of baseline at 6 h (P < 0.005). The LRP/BA mRNA value was also lower at 6 h (16% decrease, P < 0.05). HMGCR mRNA expression was unchanged. C homozygotes for the C200T polymorphism had higher LDLR/BA values than T homozygotes (P = 0.01) and although plasma LDL cholesterol (LDL-C) concentrations decreased in the postprandial period (P < 0.002), the decrease was less in C than in T homozygotes (P < 0.05). This study constitutes the first observation, to our knowledge, of postprandial changes in LDLR and LRP mRNA expression. It documents immediate effects of a fatty meal on these mRNA as well as an LRP genotype effect on LDLR mRNA and LDL-C.

Genotyping of single nucleotide substitutions.

The description of the polymerase chain reaction in 1985 caused a revolution in genetics and today molecular diagnostics is one of the leading growth areas across all disciplines of laboratory medicine. This paper reviews the principles and limitations of a number of traditional and emerging techniques for typing of single nucleotide substitutions. The techniques discussed include traditional approaches such as restriction enzyme analysis, more recent homogenous methods, such as those utilising TaqMan, fluorescence resonance energy transfer (FRET) and Scorpion probes, and high resolution melting curve analysis. Non-homogenous but highly flexible approaches such as Pyrosequencing and mass-spectrometry are also discussed. While many techniques are available, it is clear that no one approach is clearly superior. However, in terms of their many advantages and continuing developments, homogenous approaches have much to recommend them.

Evidence of reduced coronary artery disease risk for apolipoprotein epsilon2/3 heterozygotes.

BACKGROUND: Polymorphism in the gene for apolipoprotein E (apo E) influences lipid metabolism. Relative to the epsilon3 allele, the epsilon4 allele tends to increase and the epsilon2 allele tends to decrease total and serum cholesterol, but uncertainty remains concerning an influence on the risk of coronary artery disease (CAD). It is possible that the influence of apo E alleles on CAD risk is influenced by the age of subjects studied. In this study, we examine the influence of the epsilon2 and epsilon4 alleles on the risk of CAD in relatively young subjects. METHODS: We determined the apo E genotype of 564 Caucasian CAD subjects below 50 years of age presenting with symptomatic CAD, either with or without prior myocardial infarction, and documented by angiography, and 639 similarly aged Caucasian control subjects without symptomatic CAD randomly selected from the community. RESULTS: The frequency of subjects with the epsilon2/3 genotype was significantly lower in CAD subjects than controls (6 vs. 11%, P<0.01) and, relative to epsilon3/3, the epsilon2/3 genotype was associated with a significant reduction in total and LDL-cholesterol in male and female control subjects. In contrast, there was no difference in the frequency of epsilon4/4 or epsilon4/3 genotypes in CAD cases and controls (30 vs. 26%, NS), and the latter genotypes had little influence on total or LDL-cholesterol. CONCLUSION: The results indicate a beneficial effect of the epsilon2/3 genotype not only on LDL cholesterol but in decreasing the risk of CAD in Caucasians at a young age.

Hepatic lipase gene -514 C/T polymorphism and premature coronary heart disease.

BACKGROUND: A common polymorphism in the hepatic lipase (HL) gene promoter, -514C/T, affecting enzyme activity, has been associated with alterations in plasma lipoprotein levels. However a relationship with coronary heart disease (CHD) is less well documented. DESIGN AND METHODS: We studied HL -514 C/T in 562 Caucasian CHD patients aged under 50 years and in 642 Caucasian community recruited subjects without historical evidence of CHD. RESULTS: Male CHD subjects (n = 490) had a 41% carrier rate for the C to T substitution, compared with 33% in corresponding controls (n = 330), [OR = 1.42 (95% CI:1.06-1.90), P < 0.02], T allele frequencies being 0.231 and 0.177 respectively [OR = 1.39 (1.08-1.78), P < 0.01]. In male CHD subjects, the T allele was associated with higher HDL-cholesterol (HDL-C) (CC: 0.95 +/- 0.24 (SD); CT: 1.04 +/- 0.41; TT: 1.01 +/- 0.20 mmol/l, P = 0.02, ANOVA) but the trend was not significant in females. In male CHD patients the T allele was more frequently encountered in those with high (> 4.5 mmol/l) than in those with low triglycerides [68% vs. 39%, OR = 3.13 (1.54-6.67), P = 0.001]. In community control subjects, the T allele was associated with a trend to higher HDL-C levels, the significance varying between subgroups while, in males, serum total and LDL-cholesterol were significantly lower in T homozygotes than in the other two genotypes (LDL-C: 2.73 +/- 0.63 vs. 3.56 +/- 0.95 mmol/l; P = 0.01). During the course of this study, a previously unreported promoter region polymorphism was found exclusively on -514C chromosomes (-592A/G, A allele frequency 0.108, 95% CI 0.09 - 0.126). It can lead to mistyping of C as T alleles in C/T heterozygotes, resulting in overestimation of -514 T homozygotes. CONCLUSIONS: The T allele of the hepatic lipase -514 C/T polymorphism is associated with changes in plasma lipids. The superficially paradoxical predisposition to CHD in males is attributable to impairment of TG rich lipoprotein metabolism and reverse cholesterol transport.