Higher Creatinine Concentrations in Ethyl Glucuronide-Positive Urine Specimens Collected from Subjects in a Controlled Alcohol Abstinence Program: Is Serum Creatinine a Good Marker of Renal Function in Drinkers?

AIMS: The aim of this study was to examine urine creatinine concentrations in drivers submitted to controlled alcohol abstinence programs. METHODS: Urine samples (n = 32,210) were screened for ethyl glucuronide (EtG) by immunoassay during a 2-year period. Non-negatives underwent EtG and ethyl sulfate (EtS) confirmation by coupled-column Liquid Chromatography-Tandem Mass Spectrometry. Urine samples were tested for dilution by the analysis of creatinine content with <0.2 g/l indicating a dilute specimen. RESULTS: The mean urine creatinine was significantly higher in EtG positives compared to negatives (1.47 ± 0.98 vs. 1.17 ± 0.79 g/l). The difference between positives and negatives was consistent within genders and age groups (<45; ≥45). The higher urinary creatinine in EtG positives is explained by a late antidiuretic effect of alcohol. CONCLUSION: Attempts to dilute urine specimens by drinking water or other liquids before voiding are less effective for EtG/EtS compared with illicit drugs excreted in urine. If the temporary decrease in serum creatinine as a consequence of the late antidiuretic effect of alcohol is confirmed by controlled studies, serum creatinine as an indicator of kidney function should be reconsidered in drinkers.

Effects of infection control measures towards preventing SARS-CoV-2 outbreaks in a German choir boarding school from March 2020 to April 2022.

Background: Singing in a choir was associated with larger outbreaks in the beginning of the SARS-CoV-2 pandemic. Materials and methods: We report on the effect and acceptance of various infection control measures on the occurrence of SARS-CoV-2 infections in the world famous Domspatzen boys' choir from March 2020 to April 2022. Results: In addition to basic general hygiene measures, systematic rRT-PCR testing and scientifically approved concepts of distancing during singing were applied. While single infections of choir members could not be avoided, singing-related outbreaks were not observed. Until the Omicron variant emerged, potential transmission of SARS-CoV-2 in the school was limited to only one case. Incidences at the school were never higher than in the comparable general population until then. While the impact of the pandemic on daily life and singing was rated as severe, especially by staff members, most students agreed with the usefulness of protection measures and rated them as acceptable. Students viewed regular testing as the most important tool to increase safety in the school. Discussion: A bundle of infection control measures including regular testing can prevent outbreaks of SARS-CoV-2 even in the setting of choir singing. Measures are acceptable for choir members if they allow to continue with singing and performing.

Docosahexaenoic acid attenuates microglial activation and delays early retinal degeneration.

Microgliosis is a common phenomenon in neurodegenerative disorders including retinal dystrophies. We performed a detailed characterization of activated microglia in the retinoschisin (Rs1h)-deficient (Rs1h(-/Y)) mouse model of inherited retinal degeneration. To visualize and isolate microglia, we crossed Rs1h(-/Y) animals with transgenic MacGreen mice, which express green fluorescent protein under the control of the macrophage-specific csf1r promoter. Activated microglia were detected in retinal sections and whole-mounts of early postnatal MacGreen/Rs1h(-/Y) mice before the onset of overt neuronal cell death. These activated microglia contained prominent lipid droplets and analysis of the retinal lipid composition showed decreased docosahexaenoic acid (DHA) levels in Rs1h(-/Y) retinas. To establish a link between microglia activation, reduced DHA levels, and neurodegeneration, a dietary intervention study was performed. Female Rs1h(-/-) mice and their Rs1h(-/Y) litter were either subjected to a diet enriched with DHA, or a control chow lacking DHA. Supplementation with DHA enhanced photoreceptor survival and converted activated microglia to a quiescent phenotype. Furthermore, DHA, but not docosapentaenoic acid or adrenic acid reduced pro-inflammatory gene expression, migration, and lipid accumulation of cultured BV-2 microglia. We conclude that retinal DHA levels control the activity of microglia and thereby may affect the progression and extent of retinal degeneration.

High glucose, unsaturated and saturated fatty acids differentially regulate expression of ATP-binding cassette transporters ABCA1 and ABCG1 in human macrophages.

The ATP-binding cassette transporters ABCA1 and ABCG1 are highly expressed in macrophage-derived foam cells and promote reverse cholesterol efflux via biogenesis of high-density lipoproteins. The aim of this study was to analyze the direct effects of bioactive factors related to the metabolic syndrome on macrophage transcript levels of all 47 human ABC transporters. Using in vitro M-CSF predifferentiated macrophages and TaqMan low density arrays we could show that linoleic acid, palmitic acid, and high glucose levels have a major impact on ABCA1 and ABCG1 expression but do not strongly affect most other human ABC transporters. In Western blot experiments we demonstrate that ABCA1 and ABCG1 protein levels are synchronously suppressed by high glucose levels and the w6-unsaturated fatty acid linoleic acid. We conclude that metabolites associated with the metabolic syndrome enhance the formation of atherosclerotic lesions by diminishing the reverse cholesterol transport function of ABCA1 and ABCG1.

Mutations in the human ATP-binding cassette transporters ABCG5 and ABCG8 in sitosterolemia.

Phytosterolemia or Sitosterolemia is a rare autosomal recessive disorder characterized by highly elevated plasma levels of plant sterols and cholesterol as a consequence of hyperabsorption and impaired biliary secretion of sterols. The disease is caused by mutations in two half size ATP-binding cassette transporters, ABCG5 and ABCG8. We have analyzed the genomic sequence of ABCG5 and ABCG8 in five well-characterized patients with Sitosterolemia. In the first patient we found a heterozygous mutation in exon 8 of the ABCG5 gene leading to a premature termination of the protein (Arg408Ter). This German patient is the first European showing a mutation of the ABCG5 gene. In a second patient we found a novel heterozygous mutation in exon 5 of ABCG8 (c.584T>A; Leu195Gln). Both patients were heterozygous for the identified mutation, but no mutation could be identified on the other chromosome. In three further analyzed patients we found mutations in exons 7, 9 and 11 of the ABCG8 gene, respectively, of which two result in a premature termination signal for translation products. One of these patients was compound heterozygous (Trp361Ter and Arg412Ter), the other was homozygous for Trp361Ter. The third patient was homozygous for an amino acid exchange (Gly574Arg). In conclusion this report describes one novel mutation affecting a highly conserved amino acid and two previously identified mutations in the ABCG8 gene. In addition, we identified for the first time a mutation in the ABCG5 gene of a European Sitosterolemia patient.

Monoclonal antibodies for the immunotherapy of solid tumours.

More than 80% of all cancers are caused by solid malignancies. More than 90% of these tumours are of epithelial origin. The main principles in tumour treatment are surgery, radiotherapy, chemotherapy or combinations of these. Complete surgical removal of the tumour is the most effective therapy for solid malignancies. Recent advances in early cancer detection led to a higher rate of resectable primary tumours and therefore prognosis will be determined especially by metastasis. Even in early stages some tumour cells may disseminate for example into the bone marrow. If these occult metastasis get evident they are mostly incurable by surgery and often highly resistant to chemotherapy. Developing new therapeutic agents to destroy these resting cells is a major challenge for the improvement of cancer therapy in the future. Advances to reach these goals were made in immunological therapies with monoclonal antibodies (MABs). These MABs are for example directed against tumor-associated antigens (TAAs). By binding selectively on tumor cells they can activate immunological effector mechanisms, e.g. antibody dependent cell cytotoxicity (ADCC) or complement mediated lysis. Other mechanisms are the blocking of inhibiting molecules to re-activate anergic tumor infiltrating lymphocytes (TILs). Furthermore growing tumours depend on oxygen supply, i.e. on effective neovascularisation. Antibodies against VEGF are able to inhibit neovascularisation and are therefore used successfully in tumour therapy.

Comprehensive mRNA profiling of lipid-related genes in microglia and macrophages using taqman arrays.

Quantitative real-time reverse-transcription (RT)-PCR is a precise and sensitive method to measure mRNA levels over a broad dynamic range. This chapter describes the quantitative transcript analysis of 41 selected lipid-related transcripts in macrophages and microglia using a novel "Lipidomic" Taqman Array. The Taqman Array results show that (1) stimulation with the liver-X-receptor and retinoid-X-receptor ligands T0901317 and 9-cis retinoic acid induces several genes of lipid metabolism, (2) lipopolysaccharide (LPS) and interferon-g (Ifn-g) strongly repress lipid-related genes, and (3) coincubation with docosahexaenoic acid dampens the repressing effect of LPS. The method described in this chapter can be used to monitor the transcriptional response of 41 dynamic "lipid" genes simultaneously in any cell type.

Human ATP-binding cassette transporter TaqMan low-density array: analysis of macrophage differentiation and foam cell formation.

BACKGROUND: ATP-binding cassette (ABC) transporters cause various diseases and regulate many physiologic processes, such as lipid homeostasis, iron transport, and immune mechanisms. Several ABC transporters are involved in bile acid, phospholipid, and sterol transport, and their expression is itself controlled by lipids. In addition, ABC proteins mediate drug export in tumor cells and promote the development of multidrug resistance. METHODS: We created an ABC Transporter TaqMan Low-Density Array based on an Applied Biosystems 7900HT Micro Fluidic Card. We used a 2-microL reaction well with 2 ng of sample. To evaluate this method for lipidomic research and to characterize expression patterns of ABC transporters in cells relevant for atherosclerosis research, we monitored mRNA expression in human primary monocytes, in vitro-differentiated macrophages, and cells stimulated with the liver-X-receptor and retinoid-X-receptor agonists T0901317 and 9-cis retinoic acid, mimicking sterol loading. RESULTS: The method enabled simultaneous analysis of 47 human ABC transporters and the reference gene 18S rRNA in 2 replicates of 4 samples per run. CONCLUSIONS: The new system uses only 2 ng of sample and small volumes of reagent, and the precaptured primers and probes avoided labor-intensive pipetting steps. The ABC Transporter TaqMan Low-Density Array may be a useful tool to monitor dysregulated ABC transporter mRNA profiles in human lipid disorders and cancer-related multidrug resistance and to analyze the pharmacologic and metabolic regulation of ABC transporter expression important for drug development in large-scale screening approaches.

Real-time reverse transcription-PCR expression profiling of the complete human ATP-binding cassette transporter superfamily in various tissues.

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in many physiologic processes, such as lipid transport, sterol homeostasis, immune mechanisms, and drug transport, and cause various human inherited diseases. Thus, the analysis of ABC transporter mRNA expression profiles for basic research, especially in the field of lipid metabolism, for clinical diagnosis, and for monitoring of drug effects is of great interest. METHODS: We have developed a rapid, accurate, and highly sensitive real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of all 47 currently known members of the ABC transporter superfamily. Our expression analysis is based on relative quantification using a calibration curve method. With our assay, expression monitoring of a large number of RNA samples in a 384-well format with only 50 ng of total RNA is possible. RESULTS: In contrast to previous expression analyses of single ABC genes, our method allows the rapid and complete analysis of all ABC transporters in given RNA samples. We used our newly established expression panel to study the gene expression of all human ABC transporters in 20 different human tissues. As a result, we identified tissues with high transcriptional activity for ABC transporters. These organs are mainly involved in secretory function (adrenal gland), metabolic function (liver), barrier function (lung, trachea, small intestine), and tropic function (placenta, uterus). CONCLUSIONS: Our RT-PCR assay allows rapid, high-throughput transcriptional profiling of the complete ABC transporter superfamily and thus provides a new enabling tool for research, clinical diagnosis of disease, and drug testing and development.

Loss of detoxification in inflammatory bowel disease: dysregulation of pregnane X receptor target genes.

BACKGROUND & AIMS: Phase 1, phase 2, and cellular efflux transporters are critical components in intestinal barrier function against xenobiotics and bacteria. We therefore performed global gene expression profiling in patients with ulcerative colitis (UC) and Crohn's disease as well as control specimens, with a special emphasis on genes involved in detoxification and epithelial membrane integrity. METHODS: Mucosal biopsy specimens from nonaffected regions of the colon and the terminal ileum were subjected to DNA microarray analysis and pathway-related data mining. Real-time reverse-transcription polymerase chain reaction was used for verification of selected regulated candidate genes in larger inflammatory bowel disease sample numbers and intestinal cell lines. RESULTS: Several dysregulated genes were identified in both disease groups and tissues. A set of genes coordinately down-regulated in the colon of patients with UC was composed of cellular detoxification and defense genes, which are target genes for the transcription factor pregnane X receptor (PXR). Messenger RNA expression of ABCB1 (MDR1) and PXR was significantly reduced in the colon of patients with UC but was unaffected in patients with Crohn's disease. In contrast to some of its target genes, the expression of PXR was not sensitive to tumor necrosis factor alpha stimulation of intestinal cell lines. CONCLUSIONS: A disease- and tissue-specific decrease in the expression of detoxification enzymes and ABC transporters was observed, which may be explained by a loss of PXR expression. Thus, dysregulation of xenobiotic metabolism and PXR activity in the gut is likely to contribute to the pathophysiology of UC.