Gender and gut microbiota composition determine hepatic bile acid, metabolic and inflammatory response to a single fast-food meal in healthy adults.

BACKGROUND & AIMS: Regular consumption of fast-food (FF) as a form of typical Western style diet is associated with obesity and the metabolic syndrome, including its hepatic manifestation nonalcoholic fatty liver disease. Currently, it remains unclear how intermittent excess FF consumption may influence liver metabolism. The study aimed to characterize the effects of a single FF binge on hepatic steatosis, inflammation, bile acid (BA), glucose and lipid metabolism. METHODS: Twenty-five healthy individuals received a FF meal and were asked to continue eating either for a two-hour period or until fully saturated. Serum levels of transaminases, fasting BA, lipid profile, glucose and cytokine levels as well as transient elastography and controlled attenuation parameter (CAP; to assess hepatic steatosis) were analyzed before (day 0) and the day after FF binge (day 1). Feces was collected prior and after the FF challenge for microbiota analysis. RESULTS: The FF meal induced a modest increase in CAP, which was accompanied by a robust increase of fasting serum BA levels. Surprisingly, levels of cholesterol and bilirubin were significantly lower after the FF meal. Differentiating individuals with a relevant delta BA (>1 µmol/l) increase vs. individuals without (delta BA ≤1 µmol/l), identified several gut microbiota, as well as gender to be associated with the BA increase and the observed alterations in liver function, metabolism and inflammation. CONCLUSION: A single binge FF meal leads to a robust increase in serum BA levels and alterations in parameters of liver injury and metabolism, indicating a novel metabolic aspect of the gut-liver axis.

Long-lasting stability of vaccinia virus (orthopoxvirus) in food and environmental samples.

Poxviruses are known to remain infectious in the scabs of patients for months to years. The aim of this study was to investigate viral stability in storm water, food or gauze spiked with vaccinia virus strain Munich 1 (VACV M1). Storm water, storm water supplemented with either fetal calf serum (FCS) or potting soil was stored at two different temperatures (refrigerator, room temperature; 4 degrees C/25 degrees C). In addition, we analysed the viability of VACV M1 on the surface of bread, salad, sausages and gauze bandages stored at 4 degrees C. Samples were titrated in MA 104 cells and the presence of viral DNA was demonstrated by orthopoxvirus-specific PCRs. After 2 weeks, reisolation of VACV M1 from all kinds of food, bandage and water samples except for storm water supplemented with potting soil was possible. Viral DNA was detected in almost all samples by PCR. Prolonged experiments with VACV M1-spiked storm water and storm water supplemented with FCS revealed that samples kept at 4.5 degrees C are infectious for up to 166 days. Our data demonstrate that VACV M1 has a longlasting stability in water and food. The results obtained during this study should be taken into account for risk assessment calculations for poxvirus transmission. Implying that variola virus and vaccinia virus behave in a similar way, our data call for sophisticated countermeasures in cases of a variola release in biological warfare.

Post-hoc analysis on the CD14 C(-260)T promoter polymorphism and coronary heart disease.

Functional C(-260)--> T polymorphism in the promoter of the CD14 gene has been reported to be associated with coronary heart disease (CHD). The functional role of the polymorphism, however, is still a matter of debate, since several studies have not proved its effect on clinical outcomes associated with atherosclerosis. Cardiovascular-related morbidity and mortality was assessed in a post-hoc approach four years after baseline characterization of patients (male/female n = 36/32) with angiographically proven coronary heart disease. CD14 C(-260)--> T promoter genotype was determined at baseline. Seventeen out of 20 CHD patients with non-lethal cardiovascular events carried at least one T-allele. CD14 T-260 allele carriers have a 3.59-fold (95 % confidence interval: 1.11-6.75) increased risk for non-lethal cardiovascular events (Kaplan-Meier plot: log rank test p = 0.029). All patients with lethal outcomes (n = 6) were also T-allele carriers. Multivariate logistic regression analysis among CHD patients including age, established risk factors and the C(-260)--> T polymorphism as covariates and non-lethal events as a dependent variable confirmed the independent prospective effect of the T-allele on cardiovascular outcomes in this subset. Further evidence is provided for the role of CD14 C(-260)--> T promoter polymorphism as a genetic susceptibility marker of atherosclerosis in patients with an advanced clinical course of the disease. Due to the small sample size and post-hoc character of the study large-scale prospective studies that monitor patients with proven CHD are needed to confirm these findings.

A novel screening ELISA and a confirmatory Western blot useful for diagnosis and epidemiological studies of tularemia.

A novel enzyme-linked immunosorbent assay (ELISA) and a confirmatory Western blot (WB) to detect human antibodies against Francisella tularensis were evaluated. The ELISA was based on partially purified lipopolysaccharide (LPS), the WB on whole antigen of F. tularensis. Positive WB showed a typical LPS ladder. Sensitivity and specificity of the ELISA, as assessed in 104 positive sera and 1149 'normal' sera from healthy young adults, were 99.0% and 97.1% respectively. Sensitivity of the WB was close to 100%, whereas specificity was 99.6%. Antibodies against the LPS of F. tularensis were detected in four of the 'normal' sera in both ELISA and WB. The assays were further evaluated using sera of individuals from Norway, Sweden and Kosovo suspected to be infected in tularemia outbreaks. Results revealed that the combination of ELISA and WB is suitable for laboratory confirmation of tularemia as well as for large-scale epidemiological studies.

The commercially available ELISA for pancreatic elastase 1 based on polyclonal antibodies does measure an as yet unknown antigen different from purified elastase 1. Binding studies and clinical use in patients with exocrine pancreatic insufficiency.

The measurement of fecal elastase 1 concentrations by means of an ELISA based on monoclonal antibodies (mABs) highly specific for human elastase 1 (ELISA 1) has become an accepted indirect test of the exocrine pancreatic function during the last years. Its use has been demonstrated in many clinical studies including comparison with direct function tests and ERCP morphology. Recently, a new ELISA, also named "elastase 1" based on polyclonal antibodies (pABs; ELISA 2) became available. In the present investigation we performed binding studies with purified elastase 1 as well as studies on patients with exocrine insufficiency with both ELISAs. Surprisingly, the pABs on the solid phase (catcher antibodies) of ELISA 2 did not bind purified elastase 1. These antibodies seem to react with an as yet unknown antigen associated with elastase 1. Measurement of samples from patients suspected to suffer from exocrine insufficiency showed a weak correlation of both assays but higher levels in ELISA 2, resulting in false normal results even in some patients with pancreatic steatorrhea. Since the reference range used in both assays has been established using ELISA 1, ELISA 2 must be re-evaluated in comparison to direct function tests. ELISA 2 should be renamed, since obviously it does react with an antigen (antigens) different from elastase 1.

Effects of fluvastatin on biliary lipids in subjects with an elevated cholesterol saturation index.

OBJECTIVE: 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have been suggested as agents to reduce the biliary cholesterol saturation index (CSI) in duodenal bile and therefore might be supportive in primary or secondary prevention of gallstones. However, the efficiency of the therapy seems to depend on both the HMG-CoA reductase inhibitor used and the study population selected. METHODS: We therefore investigated the effect of a high-dose application of fluvastatin on biliary lipid composition in 21 subjects exhibiting mild hypercholesterolaemia and a history of current gallstones or cholecystectomy due to gallstone disease. Subjects were treated either with 40 mg fluvastatin twice per day over a 3-month period (n = 14) or with placebo (n = 7). Bile samples were aspirated during endoscopy after intravenous ceruletid stimulation before and after therapy. RESULTS: Both groups were comparable in CSI (mean +/- SD) at baseline (1.78 +/- 0.2 placebo vs. 1.97 +/- 0.4 verum). CSI significantly decreased in the verum group to 1.45 +/- 0.4 (P = 0.003) mainly due to increased phospholipid levels, whereas no difference was observed in the placebo group (1.85 +/- 0.7, n.s.). In addition, the verum group exhibited a significant reduction of hydrophobic deoxycholic acid, which has been reported to induce cholesterol crystal precipitation, and an increase of hydrophilic cholic acid. CONCLUSION: Fluvastatin might decrease the risk of cholesterol gallstone formation in patients with elevated biliary CSI during long-term treatment by reduction of biliary cholesterol saturation and percentage change in deoxycholic acid content.

The zinc finger protein 202 (ZNF202) is a transcriptional repressor of ATP binding cassette transporter A1 (ABCA1) and ABCG1 gene expression and a modulator of cellular lipid efflux.

The zinc finger gene 202 (ZNF202) located within a hypoalphalipoproteinemia susceptibility locus on chromosome 11q23 is a transcriptional repressor of various genes involved in lipid metabolism. To provide further evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia, we investigated the effect of ZNF202 expression on ATP binding cassette transporter A1 (ABCA1) and ABCG1. ABCA1 is a key regulator of the plasma high density lipoprotein pool size, whereas ABCG1 is another mediator of cellular cholesterol and phospholipid efflux in human macrophage. We demonstrate here that the full-length ZNF202m1 isoform binds to GnT repeats within the promotors of ABCA1 (-229/-210) and ABCG1 (-572/-552). ZNF202m1 expression in HepG2 cells dose-dependently repressed the promotor activities of ABCA1 and ABCG1. This transcriptional effect required the presence of the SCAN domain in ZNF202 and the functional integrity of a TATA box at position -24 of ABCA1, whereas the presence of GnT binding motifs was nonessential. The state of ZNF202 SCAN domain oligomerization affected the ability of the adjacent ZNF202 Krüppel-associated box domain to recruit the transcriptional corepressor KAP1. Overexpression of ZNF202m1 in RAW264.7 macrophages prevented the induction of ABCA1 gene expression by 20(S)OH-cholesterol and 9-cis-retinoic acid, further substantiating the interference of ZNF202 in critical elements of transcriptional activation. Finally, HDL and apoAImediated lipid efflux was significantly reduced in RAW264.7 cells stably expressing ZNF202m1. In conclusion, we have identified ABCA1 and ABCG1 as target genes for ZNF202-mediated repression and thus, provide evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia.

Complete coding sequence, promoter region, and genomic structure of the human ABCA2 gene and evidence for sterol-dependent regulation in macrophages.

Members of the human ABC transporter A subfamily have gained considerable attention based on the recent findings that ABCA1 and ABCR (ABCA4) cause familial HDL-deficiency syndromes and distinct forms of hereditary retinopathies, respectively. Here we report the complete cDNA and the genomic organization of ABCA2, another member of the human ABC A transporter subfamily. The ABCA2 coding region is 7.3 kb in size and codes for a 2436 amino acid polypeptide that bears the typical features of a full-size ABC transporter. Among the known members of the ABC A subfamily ABCA2 shares highest homology with the cholesterol-responsive transporters ABCA1 (50%) and the recently cloned ABCA7 (44%). The ABCA2 gene comprises 48 exons which are localized within a genomic region of only 21 kb. Analysis of the putative ABCA2 promoter sequence revealed potential binding sites for transcription factors that are involved in the differentiation of myeloid and neural cells. Gene expression analysis in human macrophages showed that ABCA2 mRNA is induced during cholesterol import indicating that ABCA2 is a cholesterol-responsive gene. Our results suggest a potential role for ABCA2 in macrophage lipid metabolism and neural development.

Genomic organization and characterization of the promoter of the human ATP-binding cassette transporter-G1 (ABCG1) gene.

The ATP-binding cassette transporter G1 (ABCG1) was recently identified as a regulator of macrophage cholesterol and phospholipid transport. This transporter together with ABCA1 belongs to a group of sterol-sensitive ABC proteins which are induced by lipid loading or specific oxysterols. We report here the genomic structure of ABCG1 along with the 5' flanking sequence using library screening and BLAST search analysis. The ABCG1 gene spans more than 70 kb and contains 15 exons. The exon size is between 30 and 1081 bp and the introns range in size from 137 bp to more than 45 kb. All exon-intron boundaries display the canonical GT/AG sequences. Using promoter-luciferase reporter assays in the myeloid cell lines THP-1 and RAW246.7 and the hepatoma cell line HepG2 we could demonstrate the functionality of the ABCG1 promoter and the minimal sequence requirements for gene expression. The TATA-less proximal promoter contains multiple Sp1 binding sites and a consensus sequence for sterol regulatory element binding protein.

ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport.

Excessive uptake of atherogenic lipoproteins such as modified low-density lipoprotein complexes by vascular macrophages leads to foam cell formation, a critical step in atherogenesis. Cholesterol efflux mediated by high-density lipoproteins (HDL) constitutes a protective mechanism against macrophage lipid overloading. The molecular mechanisms underlying this reverse cholesterol transport process are currently not fully understood. To identify effector proteins that are involved in macrophage lipid uptake and release, we searched for genes that are regulated during lipid influx and efflux in human macrophages using a differential display approach. We report here that the ATP-binding cassette (ABC) transporter ABCG1 (ABC8) is induced in monocyte-derived macrophages during cholesterol influx mediated by acetylated low-density lipoprotein. Conversely, lipid efflux in cholesterol-laden macrophages, mediated by the cholesterol acceptor HDL(3), suppresses the expression of ABCG1. Immunocytochemical and flow cytometric analyses revealed that ABCG1 is expressed on the cell surface and in intracellular compartments of cholesterol-laden macrophages. Inhibition of ABCG1 protein expression using an antisense strategy resulted in reduced HDL(3)-dependent efflux of cholesterol and choline-phospholipids. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified an additional set of ABC genes whose expression is regulated by cholesterol uptake or HDL(3)-mediated lipid release, suggesting a potential function for these transporters in macrophage lipid homeostasis. Our results demonstrating a regulator function for ABCG1 in cholesterol and phospholipid transport define a biologic activity for ABC transporters in macrophages.

Pancreatic exocrine function in patients with type 1 and type 2 diabetes mellitus.

Reduced exocrine pancreatic function has been observed in a high percentage of patients with type 1 diabetes in the past. There are only few data for type 2 diabetes available and they are contradictory. In this study we investigated exocrine pancreatic function in 105 controls and 114 patients with type 1 or type 2 diabetes mellitus by means of an indirect test (faecal elastase-1 concentration). This test has good sensitivity and specificity for moderate and severe pancreatic insufficiency as compared to the gold standard. Reduced faecal elastase-1 concentrations were found in 56.7% of type 1 patients, 35% of type 2 patients and 18.1% of the controls. Elastase-1 concentrations did not correlate with alcohol consumption, diabetes duration or diabetes therapy. The data found for type 1 patients correspond to those reported in earlier studies. The results for type 2 diabetics show that exocrine pancreatic function is also impaired in a high percentage in this group of patients. Pathogenetic concepts to explain these findings as consequences of diabetes complications or insulin deficiency are still under debate. Observations from autopsies and the data of the controls in this study suggest that chronic pancreatitis might be a common problem. In consequence, diabetes secondary to exocrine disease could be much more frequent than believed so far.

The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.

ATP-binding cassette transporter A1 (ABCA1) in macrophages: a dual function in inflammation and lipid metabolism?

Activated lipid-laden macrophages in the vascular wall are key modulators of the inflammatory processes underlying atherosclerosis. We demonstrate here that the ATP-binding cassette (ABC) transporter ABCA1 is induced during differentiation of human monocytes into macrophages. ABCA1 has been implicated in macrophage interleukin-1beta secretion and apoptosis. Moreover, ABCA1 mRNA and protein levels are strongly upregulated by uptake of modified LDL and downregulated by HDL(3)-mediated lipid efflux in macrophages. Mutation analysis in patients with the classical Tangier disease (TD), a monogenetic disorder characterized by hypersplenism, macrophage accumulation and deposition of cholesteryl esters in the reticuloendothelial system, low plasma HDL and premature atherosclerosis, revealed deleterious mutations in their ABCA1 gene. The localization pattern of the mutations within the ABCA1 protein appears to determine the tropism for either the reticuloendothelial system, as seen in the classical TD phenotype, or the artery wall, as in the case of HDL deficiency in the absence of splenomegaly. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified additional cholesterol-responsive genes that are induced during monocyte differentiation into macrophages. Our results indicate a dual regulatory function for ABCA1 in macrophage lipid metabolism and inflammation.

Fluorometric determination of total retinyl esters in triglyceride-rich lipoproteins.

A time-consuming sample preparation and measuring procedure is required for the quantitation of retinyl palmitate by HPLC. We developed a fluorometric method for the determination of total retinyl esters in chylomicrons, chylomicron remnants, and VLDL. This method is precise, sensitive, rapid, simple, and particularly useful for large-scale studies of postprandial lipid metabolism. Because the turbidity of postprandial lipemic samples interferes with the fluorescence measurement, all samples were incubated for 10 min with a clearing buffer containing esterase and detergents. This buffer eliminates the turbidity and hydrolyzes all retinyl esters to retinol. The fluorescence signal (excitation wavelength, 330 nm; emission wavelength, 490 nm) was linear from 0.1 mg/L up to 4 mg/L retinyl palmitate, and the CVs were 3.6% within-run and 5.1% within-series. A first application studied postprandial lipoproteins, which were first separated by ultracentrifugation and then subjected to size exclusion chromatography. Fluorescence analysis revealed that the chylomicron density fraction contains large amounts of chylomicron remnants.