Association of the habitual dietary intake with the fatty liver index and effect modification by metabotypes in the population-based KORA-Fit study.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is an emerging threat for public health with diet being a major risk factor in disease development and progression. However, the effects of habitual food consumption on fatty liver are still inconclusive as well as the proposed role of the individuals' metabolic profiles. Therefore, the aim of our study is to examine the associations between diet and NAFLD with an emphasis on the influence of specific metabotypes in the general population. METHODS: A total of 689 participants (304 men and 385 women) of the KORA-Fit (S4) survey, a follow-up study of the population-based KORA cohort study running in the Region of Augsburg, Germany, were included in this analysis. Dietary information was derived from repeated 24-h food lists and a food frequency questionnaire. The intake of energy and energy-providing nutrients were calculated using the national food composition database. The presence of fatty liver was quantified by the fatty liver index (FLI), and metabotypes were calculated using K-means clustering. Multivariable linear regression models were used for the analysis of habitual food groups and FLI; for the evaluation of macronutrients, energy substitution models were applied. RESULTS: A higher consumption of nuts and whole grains, and a better diet quality (according to Alternate Healthy Eating Index and Mediterranean Diet Score) were associated with lower FLI values, while the intake of soft drinks, meat, fish and eggs were associated with a higher FLI. The isocaloric substitution of carbohydrates with polyunsaturated fatty acids was associated with a decreased FLI, while substitution with monounsaturated fatty acids and protein showed increased FLI. Statistically significant interactions with the metabotype were observed for most food groups. CONCLUSION: The consumption of plant-based food groups, including nuts and whole grains, and diet quality, were associated with lower FLI values, whereas the intake of soft drinks and products of animal origin (meat, fish, eggs) were associated with a higher FLI. The observed statistically significant interactions with the metabotype for most food groups could help to develop targeted prevention strategies on a population-based level if confirmed in independent prospective studies.

Cathepsin S increases tau oligomer formation through limited cleavage, but only IL-6, not cathespin S serum levels correlate with disease severity in the neurodegenerative tauopathy progressive supranuclear palsy.

Limited cleavage promotes the aggregation propensity of protein tau in neurodegenerative tauopathies. Cathepsin S (CatS) is overexpressed in brains of patients suffering from tauopathies such as Alzheimer's disease (AD). Furthermore, CatS serum levels correlate with survival in the elderly. The current study investigates whether limited cleavage by CatS promotes tau aggregation, and whether CatS serum levels may correlate with disease severity in tauopathies. Oligomer formation of fluorescently labeled protein tau was monitored by single particle fluorescence spectroscopy after coincubation with CatS. Tau cleavage patterns were investigated by SDS-PAGE. For serum analyses, samples were collected from 42 patients with probable progressive supranuclear palsy (PSP) according to NINDS-PSP criteria. Disease severity was assessed by PSP rating scale (PSP-RS), PSP staging system (PSP-S) and Schwab and England Activities of Daily Living (SEADL). CatS, cystatin C (CysC) and interleukin 6 (IL-6) serum levels were determined by ELISA, ECLIA and turbidimetry, respectively. SDS-PAGE demonstrated a distinct cleavage pattern of protein tau after coincubation with CatS. Furthermore, tau oligomer formation was increased 2.4-fold (p < 0.05) after limited cleavage. Serum CatS and CysC levels did not correlate with disease severity in PSP. Of note, IL-6 correlated with PSP-S (r = 0.41; 95% CI 0.11-0.65; p = 0.008), SEADL (r = -0.37; 95% CI -0.61 to -0.06; p = 0.017) and the history and gait/midline subdomains of the PSP-RS. While CatS facilitates tau aggregation in vitro, serum levels of CatS appear not to correlate with disease severity. The observed correlation of IL-6 with disease severity warrants further investigation of inflammatory markers in PSP.

Alteration of Multiple Leukocyte Gene Expression Networks is Linked with Magnetic Resonance Markers of Prognosis After Acute ST-Elevation Myocardial Infarction.

Prognostic relevant pathways of leukocyte involvement in human myocardial ischemic-reperfusion injury are largely unknown. We enrolled 136 patients with ST-elevation myocardial infarction (STEMI) after primary angioplasty within 12 h after onset of symptoms. Following reperfusion, whole blood was collected within a median time interval of 20 h (interquartile range: 15-25 h) for genome-wide gene expression analysis. Subsequent CMR scans were performed using a standard protocol to determine infarct size (IS), area at risk (AAR), myocardial salvage index (MSI) and the extent of late microvascular obstruction (lateMO). We found 398 genes associated with lateMO and two genes with IS. Neither AAR, nor MSI showed significant correlations with gene expression. Genes correlating with lateMO were strongly related to several canonical pathways, including positive regulation of T-cell activation (p = 3.44 × 10-5), and regulation of inflammatory response (p = 1.86 × 10-3). Network analysis of multiple gene expression alterations associated with larger lateMO identified the following functional consequences: facilitated utilisation and decreased concentration of free fatty acid, repressed cell differentiation, enhanced phagocyte movement, increased cell death, vascular disease and compensatory vasculogenesis. In conclusion, the extent of lateMO after acute, reperfused STEMI correlated with altered activation of multiple genes related to fatty acid utilisation, lymphocyte differentiation, phagocyte mobilisation, cell survival, and vascular dysfunction.

Effects of a Follow-On Formula Containing Isomaltulose (Palatinose™) on Metabolic Response, Acceptance, Tolerance and Safety in Infants: A Randomized-Controlled Trial.

UNLABELLED: Effects of the dietary glycaemic load on postprandial blood glucose and insulin response might be of importance for fat deposition and risk of obesity. We aimed to investigate the metabolic effects, acceptance and tolerance of a follow-on formula containing the low glycaemic and low insulinaemic carbohydrate isomaltulose replacing high glycaemic maltodextrin. Healthy term infants aged 4 to 8 completed months (n = 50) were randomized to receive the intervention follow-on formula (IF, 2.1g isomaltulose (Palatinose™)/100mL) or an isocaloric conventional formula (CF) providing 2.1g maltodextrin/100mL for four weeks. Plasma insulinaemia 60 min after start of feeding (primary outcome) was not statistically different, while glycaemia adjusted for age and time for drinking/volume of meal 60 min after start of feeding was 122(105,140) mg/dL in IF (median, interquartile range) and 111(100,123) in CF (p = 0.01). Urinary c-peptide:creatinine ratio did not differ (IF:81.5(44.7, 96.0) vs. CF:56.8(37.5, 129),p = 0.43). Urinary c-peptide:creatinine ratio was correlated total intake of energy (R = 0.31,p = 0.045), protein (R = 0.42,p = 0.006) and fat (R = 0.40,p = 0.01) but not with carbohydrate intake (R = 0.22,p = 0.16). Both formulae were well accepted without differences in time of crying, flatulence, stool characteristics and the occurrence of adverse events. The expected lower postprandial plasma insulin and blood glucose level due to replacement of high glycaemic maltodextrin by low glycaemic isomaltulose were not observed in the single time-point blood analysis. In infants aged 4 to 8 completed months fed a liquid formula, peak blood glucose might be reached earlier than 60 min after start of feeding. Non-invasive urinary c-peptide measurements may be a suitable marker of nutritional intake during the previous four days in infants. TRIAL REGISTRATION: ClinicalTrials.gov NCT01627015.

Inflammatory role and prognostic value of platelet chemokines in acute coronary syndrome.

Activated platelets and neutrophils exacerbate atherosclerosis. Platelets release the chemokines CXCL4, CXCL4L1 and CCL5, whereas myeloperoxidase (MPO) and azurocidin are neutrophil-derived. We investigated whether plasma levels of these platelet and neutrophil mediators are affected by the acute coronary syndrome (ACS), its medical treatment, concomitant clinical or laboratory parameters, and predictive for the progression of coronary artery disease (CAD). In an observational study, the association of various factors with plasma concentrations of platelet chemokines and neutrophil mediators in 204 patients, either upon admission with ACS and 6 hours later or without ACS or CAD, was determined by multiple linear regression. Mediator release was further analysed after activation of blood with ACS-associated triggers such as plaque material. CXCL4, CXCL4L1, CCL5, MPO and azurocidin levels were elevated in ACS. CXCL4 and CCL5 but not CXCL4L1 or MPO were associated with platelet counts and CRP. CXCL4 (in association with heparin treatment) and MPO declined over 6 hours during ACS. Elevated CCL5 was associated with a progression of CAD. Incubating blood with plaque material, PAR1 and PAR4 activation induced a marked release of CXCL4 and CCL5, whereas CXCL4L1 and MPO were hardly or not altered. Platelet chemokines and neutrophil products are concomitantly elevated in ACS and differentially modulated by heparin treatment. CCL5 levels during ACS predict a progression of preexisting CAD. Platelet-derived products appear to dominate the inflammatory response during ACS, adding to the emerging evidence that ACS per se may promote vascular inflammation.

Parameters of haematology, clinical chemistry and lipid metabolism in the common marmoset and alterations under stress conditions.

Common marmosets are suitable non-human primate models for many human diseases. Standard values for blood parameters are required to evaluate physiological and pathological situations. Two studies were conducted: study I to determine standard values and study II to examine these under changed housing conditions. In study I, all parameters for clinical chemistry were similar in range for both genders with these specifics: male marmosets had significantly higher total and LDL cholesterol levels than females, whereas the mean corpuscular volume and the mean corpuscular haemoglobin were significantly lower than in females. In study II, glucose, lymphocytes and salivary cortisol were significantly lower, and faecal cortisol was increased during the change of housing conditions. In conclusion, standard values for haematology and clinical chemistry for the common marmoset were determined. Further on, parameters that are influenced by relocation stress and its importance for experimental results are described.

CD36 mRNA expression is increased in CD14+ monocytes of patients with coronary heart disease.

1. Blood-derived monocytes/macrophages within the intima of the arterial wall are the main source of inflammatory cytokines and factors contributing to lesion growth, plaque instability and thrombotic events. In the present study, we assessed the hypothesis that mRNA expression levels of candidate genes of atherosclerosis in circulating CD14(+) blood monocytes are associated with coronary heart disease (CHD). 2. We investigated mRNA expression levels using reverse transcription-polymerase chain reaction of genes involved in cholesterol uptake (macrophage scavenger receptor (MSR1), scavenger receptor class B member 1 (SRB1), lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1), CD36, LDL receptor (LDLR)), reverse cholesterol transport (apolipoprotein E (ApoE), ATP-binding cassette sub-family A member 1 (ABCA1)) and inflammation (tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), interleukin-6 (IL-6), tissue factor) in CD14(+) monocytes from 119 consecutively recruited patients and found that median CD36 mRNA expression levels were significantly increased in patients with CHD compared with controls (111 x 10(3) vs 96 x 10(3) copies/10(6) copies beta-actin, respectively; n = 79 and 40, respectively; P < 0.05), despite a high interindividual variability in gene expression. 3. A common T --> C polymorphism (rs2151916) located only 14 bp upstream of the upstream transcriptional start site did not influence CD36 expression. 4. Expression levels of the other candidate genes investigated in the present study did not show any statistically significant differences between patients with CHD and controls. 5. We conclude that CD36 mRNA expression is significantly increased in patients with CHD and may serve as an indicator of CHD burden.

HMG-CoA reductase inhibitors reduce adhesion of human monocytes to endothelial cells.

HMG-CoA reductase inhibitors (statins) are believed to reduce coronary heart disease by mechanisms in addition to their well-known cholesterol-lowering effect. We studied the effect of these drugs on monocyte cell adhesion to endothelium. Pretreatment of monocytic cells (U937, THP-1, human CD14(+) monocytes) with 0.01-10 microM concentrations of atorvastatin, cerivastatin, or simvastatin significantly reduced cell adhesion to endothelium. In contrast, pretreatment of endothelium with statins did not affect adhesion of monocytes. Adhesion of monocytes to vascular cell adhesion molecule-1-coated dishes was reduced by these drugs. Cerivastatin also reduced PMA induction of NF-kappaB. Since monocyte adhesion to endothelium is an early event in atherogenesis, treatment with statins in prevention of coronary heart disease may have additional salutary effects to lowering of plasma LDL cholesterol. Our results indicate that the reduction of monocyte adhesion by HMG-CoA reductase inhibitors may be considered as a class effect.

The influence of organ temperature on hepatic ischemia-reperfusion injury: a systematic analysis.

BACKGROUND: Although hepatic ischemia-reperfusion (I/R) injury can be reduced by cooling of the ischemic organ, a systematic in vivo analysis of the influence of organ temperature in I/R injury is missing. The aim of this study was to systematically investigate the impact of defined temperatures of the ischemic liver tissue on microvascular I/R injury. METHODS: Ischemia of the left liver lobe was induced in C57BL/6 mice for 90 min. The ischemic lobe was placed in a polyethylene well and the temperature was adjusted to 37 degrees C, 26 degrees C, 15 degrees C, and 4 degrees C by superfusion with cooled/warmed saline solution. The ischemia groups (n=7 each) were compared with a sham-operated group (n=7). The sinusoidal perfusion index and the number of leukocytes firmly adherent to the endothelium of postsinusoidal venules were assessed using intravital fluorescence microscopy at 30 min, 120 min, and 240 min of reperfusion, respectively. At the end of the experiment, serum activities of the liver enzymes aspartate aminotransferase/alanine aminotransferase were determined, and tissue specimens were examined by electron microscopy. RESULTS: Core body temperature did not differ significantly between the groups. In the 37 degrees C group, the sinusoidal perfusion index was significantly reduced and the number of adherent leukocytes was significantly increased compared with the sham group. In all hypothermia groups, however, the microcirculatory parameters did not differ from the sham group. Serum activities of aspartate aminotransferase/alanine aminotransferase were significantly increased and hepatocellular integrity was severely affected in the 37 degrees C group as compared with all other groups. CONCLUSIONS: These findings demonstrate that in the mouse liver the known protective effect of hypothermia is already encountered at 26 degrees C. Further reduction of temperature did not generate additional protection from I/R injury.

Fluorescence-based detection of the CETP TaqIB polymorphism: false positives with the TaqMan-based exonuclease assay attributable to a previously unknown gene variant.

BACKGROUND: Previous studies have shown an association between the TaqIB polymorphism of the cholesteryl ester transfer protein (CETP) gene with plasma CETP and HDL concentrations and the progression of coronary artery disease (CAD). The aim of the present study was to determine the performance of two new fluorescence-based detection systems in the analysis of the TaqIB genotype. METHODS: CAD patients (n = 150) with known TaqIB genotype, as determined by restriction fragment length polymorphism (RFLP) analysis, were selected, including three groups of 50 patients, carrying the B1/1, B1/2, and B2/2 genotypes, respectively. The genotypes were also analyzed by fluorescence-based allele-specific TaqMan PCR and melting curve analysis (LightCycler). In addition, DNA sequencing was applied. RESULTS: The TaqIB genotypes obtained by fluorescence analysis corresponded to those determined by RFLP analysis with the exception of three heterozygous patients (B1/2), who were misclassified as homozygous B2 carriers with the TaqMan system. Melting curve analysis of these samples demonstrated an additional melting point at 59.1 degrees C, which was also found in four patients homozygous for the B1 allele. DNA sequencing revealed a previously unknown C270T nucleotide exchange in intron 1 of the CETP gene, only nine base pairs from the TaqIB site. CONCLUSIONS: Determination of the TaqIB polymorphism with the TaqMan system led to misclassifications because of a previously unknown C270T polymorphism of the CETP gene. The base substitution was detected with the LightCycler because of the occurrence of an additional melting point. Our data indicate the importance of thorough evaluation of new gene analysis systems before using them on a routine basis.

Ischemia at 4 degrees C: a novel mouse model to investigate the effect of hypothermia on postischemic hepatic microcirculatory injury.

Hypothermia of the ischemic organ at 4 degrees C protects hepatic microcirculation from ischemia-reperfusion (IR) injury. The effect of hypothermia during ischemia was investigated in animal models using liver transplantation and storage of the harvested organ in cold preservation solutions. No investigation of the isolated influence of hypothermia at 4 degrees C of the ischemic organ on hepatic IR injury exists, due to the lack of an appropriate animal model. Therefore, the aim of our present study was to develop such a model using intravital video fluorescence microscopy (IVM). In C57BL/6 mice, a reversible isolated ischemia of the left liver lobe was induced for 90 min, followed by 240 min of reperfusion. The temperature of the ischemic organ was adjusted to either 4 degrees C or 37 degrees C by superfusion with 0.9% NaCl. Sham-operated animals without IR served as controls. The hepatic microcirculation was analyzed using IVM at 30 min and 240 min after reperfusion by quantifying sinusoidal perfusion and leukocyte-endothelial cell interaction in postsinusoidal venules. At the end of the experiment, blood and tissue samples were taken for measurement of liver enzyme activities and light and electron microscopy. Mean arterial pressure and body temperature were kept constant throughout the experiment, while the temperature of the ischemic liver lobe was adjusted to predefined levels. After normothermic ischemia, hepatic microvascular perfusion was significantly impaired compared with sham-operated animals. Perfusion failure was significantly reduced in hypothermic livers and did not differ from livers of the sham-group. Liver enzyme activities in the normotherimic group were significantly higher than in the sham and hypothermic groups. Light and electron microscopy revealed severe histological alterations at 37 degrees C ischemia, whereas at 4 degrees C ischemia only minimal lesions were encountered. Our novel model allows for isolated adjustment of ischemic liver lobe temperature without changing body temperature and systemic macrohemodynamic parameters. Hypothermia at 4 degrees C largely attenuates postischemic microvascular perfusion injury of the liver.

Tissue endothelin-converting enzyme activity correlates with cardiovascular risk factors in coronary artery disease.

BACKGROUND: Endothelin-converting enzymes (ECEs) are the key enzymes in endothelin-1 (ET-1) generation. However, their pathophysiological role in patients with cardiovascular disease remains elusive. METHODS AND RESULTS: Vascular reactivity to big endothelin-1 (bigET-1; 10(-9) to 10(-7) mol/L) and ET-1 (10(-9) to 10(-7) mol/L) were examined in the internal mammary artery (IMA, n=33) and saphenous vein (SV, n=27) of patients with coronary artery disease with identified cardiovascular risk factors. Vascular ECE activity was determined by conversion of exogenously added bigET-1 to ET-1. Tissue contents of bigET-1 and ET-1 were measured by radioimmunoassay. In addition, the effects of LDL and oxidized LDL on ECE-1 protein levels were determined by Western blot analysis in human IMA endothelial cells. In the IMA, vascular ECE activity showed an inverse correlation with serum LDL levels (r=-0.76; P<0.01) and systolic and diastolic blood pressure and a positive correlation with fibrinogen (r=0.58; P<0.05). In the SV, fibrinogen was the only parameter to be correlated with vascular ECE activity. Vascular tissue content of bigET-1 was attenuated in the IMA of patients with hyperfibrinogenemia but increased in patients with elevated systolic blood pressure and increased serum LDL levels (P<0.05). Most interestingly, LDL and oxidized LDL downregulated ECE-1 protein levels in human IMA endothelial cells (P<0.05). CONCLUSIONS: These data demonstrate, for the first time, that vascular ECE activity is (1) inversely correlated with serum LDL levels and blood pressure and (2) positively associated with fibrinogen in human vascular tissue. Hence, ECE-1 activity may modulate cardiovascular risk in patients with coronary artery disease.

Alpha-tocopherol down-regulates scavenger receptor activity in macrophages.

The aim of this study was to investigate the effect of alpha-tocopherol on scavenger receptor (SR) activity, SR class A (SR-A) mRNA expression and transcriptional regulation in macrophages. Scavenger receptor activity was determined quantitatively by uptake of DiI-acLDL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein) in rabbit peritoneal macrophages and human monocytes/macrophages in the presence and absence of different tocopherol homologues. SR-A mRNA expression was determined by Northern blotting, the activity of the transcription factor activator protein-1 (AP-1) by electrophoretic mobility shift assay. We could demonstrate that alpha-tocopherol down-regulates scavenger receptor activity in macrophages in a dose-dependent manner. Scavenger receptor activity was reduced by 13, 16, 18 and 24% in the presence of 1, 5, 10 and 50 microM alpha-tocopherol, respectively. This effect was associated with a reduced SR-A mRNA expression and activity of AP-1 binding transcription factors in the presence of alpha-tocopherol. The activity of scavenger receptors in human monocyte derived macrophages incubated with 100 microM alpha-tocopherol for 15 days was reduced up to 60%. Interestingly, gamma-tocopherol, which is a homologue of alpha-tocopherol with a comparable antioxidative capacity, showed only a weak suppression of SR activity, SR-A expression and AP-1 activity. Our observations point to the conclusion that the reduction of SR-A expression and activity in presence of alpha-tocopherol is possibly related to its direct action on cell signaling.

Scavenger receptor activity is increased in macrophages from rabbits with low atherosclerotic response: studies in normocholesterolemic high and low atherosclerotic response rabbits.

We have previously described 2 strains of New Zealand White rabbits with a high (HAR) or low (LAR) atherosclerotic response to hypercholesterolemia. In the present study, we focused on class A scavenger receptor (SR-A) activity and ApoE expression in macrophages from both rabbit strains. These parameters play a crucial role in maintaining cholesterol homeostasis in the arterial wall and may be involved in the development of atherosclerosis. SR activity, as measured by uptake of DiI-labeled acetylated LDL, was significantly higher in macrophages from LAR rabbits (2177+/-253 ng/mg cell protein) than in macrophages from HAR rabbits (1153+/-200 ng/mg cell protein). The higher SR activity was caused by a greater number of SRs (apparent Vmax, 4100 ng/mg in LAR and 1980 ng/mg in HAR rabbits). The high SR activity in macrophages from LAR rabbits was associated with a significantly higher expression of SR-A mRNA compared with macrophages from HAR rabbits. However, the latter finding could not be explained by differences in the activity of transcription factor-activating protein 1 (AP-1), which was comparable in macrophages from both strains of rabbits. Because under certain circumstances SR-A mRNA expression is regulated in parallel with ApoE expression, we also evaluated this parameter. Although ApoE mRNA was 74% higher in macrophages from LAR rabbits, the difference did not reach statistical significance. In conclusion, the increased expression of SR-A in macrophages in the presence of adequate amounts of ApoE may play a role in attenuating atherosclerosis in LAR rabbits.

[Genetic factors in the development of atherosclerosis]. / Genetische Faktoren der Atheroskleroseentstehung.

Atherosclerosis is a complex multifactorial disease of the arterial wall, dependent on genetic disposition and multiple other risk factors. There are probably several candidate genes, that determine the individual susceptibility of the vessel wall to develop atherosclerosis. In recent years, a growing number of gene polymorphisms, associated with an elevated risk of myocardial infarction, has been identified. These genes and gene clusters play a crucial role in lipid metabolism, regulation of blood pressure and clotting. In contrast to rare monogenetic diseases with severe clinical signs and symptoms (e.g. familial hypercholesterolemia), genetic polymorphisms are relatively frequent. Due to their frequency, there is a high probability that one individual carries several alleles predisposing to coronary heart disease. Genetic polymorphisms become clinically important by interacting with lifestyle, environmental factors or endogenous metabolic disorders. We have recently established an animal model of rabbits, which may prove useful in the search for new genes predisposing to or protecting from atherosclerosis. Rabbits with high atherosclerotic response (HAR) show more than 70% atherosclerotic involvement of the aorta when fed a high cholesterol diet. In contrast, rabbits with low atherosclerotic response (LAR) show less than 20% atherosclerosis in spite of comparably high plasma cholesterol levels. Preliminary studies indicate that macrophages of LAR rabbits have a high scavenger receptor activity and high apolipoprotein E expression and thus appear to be very efficient in uptake and elimination of modified lipoproteins. This may result in a more efficient removal of cholesterol from the arterial wall and thus protect the animals from developing atherosclerosis. Today we are only at the beginning of understanding the complexity of gene interaction in atherosclerosis. Further identification of genetic factors of atherosclerosis will no doubt lead to a more efficient and economic prevention of coronary heart disease in the future.