Comprehensive analysis of the ErbB receptor family in pediatric nervous system tumors and rhabdomyosarcoma.

BACKGROUND: There is a paucity of knowledge regarding pediatric biomarkers, including the relevance of ErbB pathway aberrations in pediatric tumors. We investigated the occurrence of ErbB receptor aberrations across different pediatric malignancies, to identify patterns of ErbB dysregulation and define biomarkers suitable for patient enrichment in clinical studies. PROCEDURE: Tissue samples from 297 patients with nervous system tumors and rhabdomyosarcoma were analyzed for immunohistochemical expression or gene amplification of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). Exploratory analyses of HER3/HER4 expression, and mRNA expression of ErbB receptors/ligands (NanoString) were performed. Assay validation followed general procedures, with additional validation to address Clinical Laboratory Improvement Amendments (CLIA) requirements. RESULTS: In most tumor types, samples with high ErbB receptor expression were found with heterogeneous distribution. We considered increased/aberrant ErbB pathway activation when greater than or equal to two EGFR/HER2 markers were simultaneously upregulated. ErbB pathway dysregulation was identified in ∼20%-30% of samples for most tumor types (medulloblastoma/primitive neuroectodermal tumors 31.1%, high-grade glioma 27.1%, neuroblastoma 22.7%, rhabdomyosarcoma 23.1%, ependymoma 18.8%), 4.2% of diffuse intrinsic pontine gliomas, and no recurrent or refractory low-grade astrocytomas. In medulloblastoma/primitive neuroectodermal tumors and neuroblastoma, this was attributed mainly to high EGFR polysomy/HER2 amplification, whereas EGFR gene amplification was observed in some high-grade glioma samples. EGFR/HER2 overexpression was most prevalent in ependymoma. CONCLUSIONS: Overexpression and/or amplification of EGFR/HER2 were identified as potential enrichment biomarkers for clinical trials of ErbB-targeted drugs.

Influence of TCF7L2 gene variants on the therapeutic response to the dipeptidylpeptidase-4 inhibitor linagliptin.

AIMS/HYPOTHESIS: Individuals carrying variants of the transcription factor 7-like 2 gene (TCF7L2) are at increased risk for type 2 diabetes. These metabolic genetic risk factors have been linked to diminished pancreatic islet-cell responsiveness to incretins, thus pharmacological interventions aimed at amplifying endogenous incretin biology may be affected. However, clinical evidence from randomised controlled trials so far is lacking. We investigated the influence of TCF7L2 risk alleles on the response to treatment with the dipeptidylpeptidase-4 (DPP-4) inhibitor linagliptin from four 24 week, phase III, placebo-controlled trials. METHODS: Pharmacogenomic samples and clinical data were available from 961 patients with type 2 diabetes. Whole-blood DNA samples were genotyped for TCF7L2 single-nucleotide polymorphisms in conjunction with assessments of 24 week changes in HbA1c. RESULTS: Linagliptin lowered HbA1c meaningfully in all three genotypes of rs7903146 (non-risk variant carriers CC [n = 356]: -0.82% [-9.0 mmol/mol], p < 0.0001; heterozygous CT [n = 264]: -0.77% [-8.4 mmol/mol], p < 0.0001; homozygous risk variant carriers TT [n = 73]: -0.57% [-6.2 mmol/mol], p < 0.0006). No significant treatment differences were seen between CC and CT patients, although HbA1c response was reduced in TT compared with CC patients (~0.26% [~2.8 mmol/mol], p = 0.0182). CONCLUSIONS/INTERPRETATION: Linagliptin significantly improved hyperglycaemia in patients with type 2 diabetes both with and without the TCF7L2 gene diabetes risk alleles. However, differences in treatment response were observed, indicating that diabetes susceptibility genes may be an important contributor to the inter-individual variability of treatment response.

Circulating biomarkers and progression of idiopathic pulmonary fibrosis: data from the INMARK trial.

Background: We used data from the INMARK trial to investigate associations between circulating biomarkers of extracellular matrix (ECM) turnover, inflammation and epithelial dysfunction and disease progression in subjects with idiopathic pulmonary fibrosis (IPF). Methods: Subjects with IPF and forced vital capacity (FVC) ≥80% predicted were randomised 1:2 to receive nintedanib 150 mg twice daily or placebo for 12 weeks followed by open-label nintedanib for 40 weeks. Associations between baseline biomarker levels and the proportion of subjects with disease progression (decline in FVC ≥10% predicted or death) over 52 weeks were assessed in subjects randomised to placebo using logistic regression. Associations between baseline demographic/clinical characteristics and biomarker levels and disease progression over 52 weeks were analysed using multivariate models. Results: Of 230 subjects who received placebo for 12 weeks then open-label nintedanib for 40 weeks, 70 (30.4%) had disease progression over 52 weeks. Baseline levels of CRPM (C-reactive protein (CRP) degraded by matrix metalloproteinase (MMP)-1/8), C3M (collagen 3 degraded by MMP-9), CRP, KL-6 (Krebs von den Lungen-6) and SP-D (surfactant protein D) were not significantly associated with disease progression over 52 weeks in analyses corrected for multiple comparisons. In models including only baseline demographic/clinical characteristics, 61.2-64.2% of subjects were correctly classified as having or not having disease progression over 52 weeks. When both demographic/clinical characteristics and biomarker levels were included in the models, 50.0-64.5% of the test set were correctly classified. Conclusions: Among subjects with IPF and preserved FVC, multivariate models based on demographic/clinical characteristics and biomarker levels at baseline did not provide an accurate prediction of which patients would progress.

An exploratory study investigating biomarkers associated with autoimmune pulmonary alveolar proteinosis (aPAP).

Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare lung disorder involving production of autoantibodies against endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF). This study aimed to identify biomarkers that could be used to monitor for aPAP, particularly in patients treated with anti-GM-CSF antibodies. This was an exploratory, prospective, observational, single-center study. Pre-specified biomarkers were evaluated between baseline and Day 120 in serum/plasma, whole blood, sputum and exhaled breath condensate from patients with aPAP, healthy volunteers, and patients with chronic obstructive pulmonary disease (COPD) and asthma (not treated with anti-GM-CSF and with no evidence of aPAP). Pulmonary function tests were also performed. Overall, 144 individuals were enrolled (aPAP: n = 34, healthy volunteers: n = 24, COPD: n = 40 and asthma: n = 46). Plasma GM-CSF levels were lower, and Krebs von den Lungen 6 and GM-CSF autoantibody ranges were higher, in patients with aPAP compared with other populations. Surfactant proteins-A and -D, lactate dehydrogenase and carcinoembryonic antigen ranges partially or completely overlapped across populations. Most plasma biomarkers showed high sensitivity and specificity for detection of aPAP; GM-CSF and GM-CSF autoantibody concentrations demonstrated equivalent sensitivity for differentiating aPAP. In addition to characteristic GM-CSF autoantibodies, assessment of plasma GM-CSF may identify individuals at risk of developing aPAP.Trial registration: EudraCT, 2012-003475-19. Registered 23 July 2012- https://eudract.ema.europa.eu/ .

FOOTPRINTS study protocol: rationale and methodology of a 3-year longitudinal observational study to phenotype patients with COPD.

INTRODUCTION: A better understanding is needed of the different phenotypes that exist for patients with chronic obstructive pulmonary disease (COPD), their relationship with the pathogenesis of COPD and how they may affect disease progression. Biomarkers, including those associated with emphysema, may assist in characterising patients and in predicting and monitoring the course of disease. The FOOTPRINTS study (study 352.2069) aims to identify biomarkers associated with emphysema, over a 3-year period. METHODS AND ANALYSIS: The FOOTPRINTS study is a prospective, longitudinal, multinational (12 countries), multicentre (51 sites) biomarker study, which has enrolled a total of 463 ex-smokers, including subjects without airflow limitation (as defined by the 2015 Global Initiative for Chronic Obstructive Lung Disease (GOLD) strategy report), patients with COPD across the GOLD stages 1-3 and patients with COPD and alpha1-antitrypsin deficiency. The study has an observational period lasting 156 weeks that includes seven site visits and additional phone interviews. Biomarkers in blood and sputum, imaging data (CT and magnetic resonance), clinical parameters, medical events of special interest and safety are being assessed at regular visits. Disease progression based on biomarker values and COPD phenotypes are being assessed using multivariate statistical prediction models. ETHICS AND DISSEMINATION: The study protocol was approved by the authorities and ethics committees/institutional review boards of the respective institutions where applicable, which included study sites in Belgium, Canada, Denmark, Finland, Germany, Japan, Korea, Poland, Spain, Sweden, UK and USA; written informed consent has been obtained from all study participants. Ethics committee approval was obtained for all participating sites prior to enrolment of the study participants. The study results will be reported in peer-reviewed publications. TRIAL REGISTRATION NUMBER: NCT02719184.

Comparison of normalization methods for Illumina BeadChip HumanHT-12 v3.

BACKGROUND: Normalization of microarrays is a standard practice to account for and minimize effects which are not due to the controlled factors in an experiment. There is an overwhelming number of different methods that can be applied, none of which is ideally suited for all experimental designs. Thus, it is important to identify a normalization method appropriate for the experimental setup under consideration that is neither too negligent nor too stringent. Major aim is to derive optimal results from the underlying experiment. Comparisons of different normalization methods have already been conducted, none of which, to our knowledge, comparing more than a handful of methods. RESULTS: In the present study, 25 different ways of pre-processing Illumina Sentrix BeadChip array data are compared. Among others, methods provided by the BeadStudio software are taken into account. Looking at different statistical measures, we point out the ideal versus the actual observations. Additionally, we compare qRT-PCR measurements of transcripts from different ranges of expression intensities to the respective normalized values of the microarray data. Taking together all different kinds of measures, the ideal method for our dataset is identified. CONCLUSIONS: Pre-processing of microarray gene expression experiments has been shown to influence further downstream analysis to a great extent and thus has to be carefully chosen based on the design of the experiment. This study provides a recommendation for deciding which normalization method is best suited for a particular experimental setup.

The transcriptional programme of contact-inhibition.

Proliferation of non-transformed cells is regulated by cell-cell contacts, which are referred to as contact-inhibition. Vice versa, transformed cells are characterised by a loss of contact-inhibition. Despite its generally accepted importance for cell-cycle control, little is known about the intracellular signalling pathways involved in contact-inhibition. Unravelling the molecular mechanisms of contact-inhibition and its loss during tumourigenesis will be an important step towards the identification of novel target genes in tumour diagnosis and treatment. To better understand the underlying molecular mechanisms we identified the transcriptional programme of contact-inhibition in NIH3T3 fibroblast using high-density microarrays. Setting the cut off: >or=1.5-fold, P or=2-fold, P

Acute and repeated flibanserin administration in female rats modulates monoamines differentially across brain areas: a microdialysis study.

INTRODUCTION: Hypoactive sexual desire disorder (HSDD) is defined as persistent lack of sexual fantasies or desire marked by distress. With a prevalence of 10% it is the most common form of female sexual dysfunction. Recently, the serotonin-1A (5-HT(1A)) receptor agonist and the serotonin-2A (5-HT(2A)) receptor antagonist flibanserin were shown to be safe and efficacious in premenopausal women suffering from HSDD in phase III clinical trials. AIM: The current study aims to assess the effect of flibanserin on neurotransmitters serotonin (5-HT), norepinephrine (NE), dopamine (DA), glutamate, and gamma-aminobutyric acid (GABA) in brain areas associated with sexual behavior. METHODS: Flibanserin was administered to female Wistar rats (280-350 g). Microdialysis probes were stereotactically inserted into the mPFC, NAC, or MPOA, under isoflurane anesthesia. The extracellular levels of neurotransmitters were assessed in freely moving animals, 24 hours after the surgery. MAIN OUTCOME MEASURES: Dialysate levels of DA, NE, and serotonin from medial prefrontal cortex (mPFC), nucleus accumbens (NAC), and hypothalamic medial preoptic area (MPOA) from female rats. RESULTS: Acute flibanserin administration decreased 5-HT and increased NE levels in all tested areas. DA was increased in mPFC and MPOA, but not in the NAC. Basal levels of NE in mPFC and NAC and of DA in mPFC were increased upon repeated flibanserin administration, when compared to vehicle-treated animals. The basal levels of 5-HT were not altered by repeated flibanserin administration, but basal DA and NE levels were increased in the mPFC. Glutamate and GABA levels remained unchanged following either repeated or acute flibanserin treatment. CONCLUSIONS: Systemic administration of flibanserin to female rats differentially affects the monoamine systems of the brain. This may be the mechanistic underpinning of flibanserin's therapeutic efficacy in HSDD, as sexual behavior is controlled by an intricate interplay between stimulatory (catecholaminergic) and inhibitory (serotonergic) systems.

High-dimensional Cox models: the choice of penalty as part of the model building process.

The Cox proportional hazards regression model is the most popular approach to model covariate information for survival times. In this context, the development of high-dimensional models where the number of covariates is much larger than the number of observations (p>>n) is an ongoing challenge. A practicable approach is to use ridge penalized Cox regression in such situations. Beside focussing on finding the best prediction rule, one is often interested in determining a subset of covariates that are the most important ones for prognosis. This could be a gene set in the biostatistical analysis of microarray data. Covariate selection can then, for example, be done by L(1)-penalized Cox regression using the lasso (Tibshirani (1997). Statistics in Medicine 16, 385-395). Several approaches beyond the lasso, that incorporate covariate selection, have been developed in recent years. This includes modifications of the lasso as well as nonconvex variants such as smoothly clipped absolute deviation (SCAD) (Fan and Li (2001). Journal of the American Statistical Association 96, 1348-1360; Fan and Li (2002). The Annals of Statistics 30, 74-99). The purpose of this article is to implement them practically into the model building process when analyzing high-dimensional data with the Cox proportional hazards model. To evaluate penalized regression models beyond the lasso, we included SCAD variants and the adaptive lasso (Zou (2006). Journal of the American Statistical Association 101, 1418-1429). We compare them with "standard" applications such as ridge regression, the lasso, and the elastic net. Predictive accuracy, features of variable selection, and estimation bias will be studied to assess the practical use of these methods. We observed that the performance of SCAD and adaptive lasso is highly dependent on nontrivial preselection procedures. A practical solution to this problem does not yet exist. Since there is high risk of missing relevant covariates when using SCAD or adaptive lasso applied after an inappropriate initial selection step, we recommend to stay with lasso or the elastic net in actual data applications. But with respect to the promising results for truly sparse models, we see some advantage of SCAD and adaptive lasso, if better preselection procedures would be available. This requires further methodological research.

The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor lovastatin reduces severity of L-DOPA-induced abnormal involuntary movements in experimental Parkinson's disease.

Chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment of Parkinson's disease (PD) often leads to debilitating involuntary movements, termed L-DOPA-induced dyskinesia (LID), about which the rodent analog, the abnormal involuntary movements (AIMs), has been associated consistently with an activation of the Ras-extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase signaling pathway. Previous studies have shown that lovastatin, a specific inhibitor of the rate-limiting enzyme in cholesterol biosynthesis, can also inhibit Ras isoprenylation and activity and subsequently the phosphorylation of ERK1/2 (pERK1/2). We hypothesized that lovastatin treatment-commenced previous L-DOPA exposure could reduce AIM incidence and severity in the 6-hydroxydopamine (6-OHDA) rat model of PD by secondarily preventing the L-DOPA/Benserazide-induced increase in pERK1 levels. The lovastatin-L-DOPA/Benserazide-treated 6-OHDA animals displayed less severe rotational behavior as well as a dramatic reduction in AIM severity than the L-DOPA/Benserazide-treated ones. Such lower AIM severity was associated with a decrease in L-DOPA-induced increase in the following: (1) striatal pERK1 and (2) DeltaFosB levels, and (3) theta/alpha oscillations of substantia nigra pas reticulata (SNr) neurons as well as (4) a normalization of SNr firing frequency. Those results strongly suggest that lovastatin might represent a treatment option for managing LID in PD.

Biomarkers of extracellular matrix turnover in patients with idiopathic pulmonary fibrosis given nintedanib (INMARK study): a randomised, placebo-controlled study.

BACKGROUND: A hallmark of idiopathic pulmonary fibrosis is the excess accumulation of extracellular matrix in the lungs. Degradation of extracellular matrix generates free-circulating protein fragments called neoepitopes. The aim of the INMARK trial was to investigate changes in neoepitopes as predictors of disease progression in patients with idiopathic pulmonary fibrosis and the effect of nintedanib on these biomarkers. METHODS: In this randomised, double-blind, placebo-controlled trial, patients with a diagnosis of idiopathic pulmonary fibrosis within the past 3 years and forced vital capacity (FVC) of 80% predicted or higher were eligible to participate. Patients were recruited from hospitals, private practices, clinical research units, and academic medical centres. Patients were randomly assigned (1:2) with the use of a pseudo-random number generator to receive oral nintedanib 150 mg twice a day or placebo for 12 weeks in a double-blind fashion, followed by open-label nintedanib for 40 weeks. The primary endpoint was the rate of change in C-reactive protein (CRP) degraded by matrix metalloproteinases 1 and 8 (CRPM) from baseline to week 12 in the intention-to-treat population. The trial has been completed and is registered with ClinicalTrials.gov, number NCT02788474, and with the European Clinical Trials Database, number 2015-003148-38. FINDINGS: Between June 27, 2016, and May 15, 2017, 347 patients were randomly assigned to the nintedanib group (n=116) or to the placebo group (n=231). One patient from the placebo group was not treated owing to a randomisation error. At baseline, mean FVC was 97·5% (SD 13·5) predicted. In the double-blind period, 116 patients received nintedanib and 230 patients received placebo. The rate of change in CRPM from baseline to week 12 was -2·57 × 10-3 ng/mL/month in the nintedanib group and -1·90 × 10-3 ng/mL/month in the placebo group (between-group difference -0·66 × 10-3 ng/mL/month [95% CI -6·21 × 10-3 to 4·88 × 10-3]; p=0·8146). The adjusted rate of change in FVC over 12 weeks was 5·9 mL in the nintedanib group and -70·2 mL in the placebo group (difference 76·1 mL/12 weeks [31·7 to 120·4]). In patients who received placebo for 12 weeks followed by open-label nintedanib, rising concentrations of CRPM over 12 weeks were associated with disease progression (absolute decline in FVC ≥10% predicted or death) over 52 weeks. In the double-blind period, serious adverse events were reported in eight (7%) patients given nintedanib and 18 (8%) patients given placebo. Grade 3 diarrhoea was reported in two (2%) patients in the nintedanib group and two (1%) patients in the placebo group. No patients had grade 4 diarrhoea. INTERPRETATION: In patients with idiopathic pulmonary fibrosis and preserved lung function, treatment with nintedanib versus placebo for 12 weeks did not affect the rate of change in CRPM but was associated with a reduced rate of decline in FVC. These results suggest that change in CRPM is not a marker of response to nintedanib in patients with idiopathic pulmonary fibrosis. FUNDING: Boehringer Ingelheim.

The histone acetyltransferase hMOF is frequently downregulated in primary breast carcinoma and medulloblastoma and constitutes a biomarker for clinical outcome in medulloblastoma.

Loss of H4 lysine 16 (H4K16) acetylation was shown to be a common feature in human cancer. However, it remained unclear which enzyme is responsible for the loss of this modification. Having recently identified the histone acetyltransferase human MOF (hMOF) to be required for bulk H4K16 acetylation, here we examined the involvement of hMOF expression and H4K16 acetylation in breast cancer and medulloblastoma. Analysis of a recent mRNA expression profiling study in breast cancer (n = 100 cases) and an array-CGH screen in medulloblastomas (n = 102 cases), revealed downregulation in 40% and genomic loss in 11% of cases, respectively. We investigated hMOF protein expression as well as H4K16 acetylation in large series of primary breast carcinomas (n = 298) and primary medulloblastomas (n = 180) by immunohistochemistry. In contrast to nontransformed control tissues, significant fractions of both primary breast carcinomas and medulloblastomas showed markedly reduced hMOF mRNA and protein expression. In addition, hMOF protein expression tightly correlated with acetylation of H4K16 in all tested samples. For medulloblastoma, downregulation of hMOF protein expression was associated with lower survival rates identifying hMOF as an independent prognostic marker for clinical outcome in univariate as well as multivariate analyses.

Molecular fingerprinting and autocrine growth regulation of endothelial cells in a murine model of hepatocellular carcinoma.

In a mouse model of hepatocellular carcinogenesis, highly vascularized tumors develop through two distinct morphologic phases of neovascularization. We show that increased vascular caliber occurs first, followed by extensive vessel sprouting in late-stage carcinomas. To define molecular pathways in tumor neovascularization, endothelial cells were directly purified from normal liver and advanced tumors. Gene expression profiling experiments were then designed to identify genes enriched in the vascular compartment. We report that Cathepsin S is the major protease specifically overexpressed during vessel sprouting. We also show that the CC chemokines CCL2 and CCL3 are secreted by neovessels and stimulate proliferation through their cognate receptors in an autocrine fashion. This suggests that chemokine signaling represents the most prominent signaling pathway in tumor-associated endothelial cells and directly regulates vessel remodeling. Furthermore, high angiogenic activity is associated with attenuated lymphocyte extravasation and correlates with expression of the immunomodulatory cytokine interleukin 10. This is the first comprehensive study addressing liver-specific vascular changes in a murine autochthonous tumor model. These novel insights into liver angiogenesis infer an environmental control of neovascularization and have important implications for the design of antiangiogenic therapies.

Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays.

Gene expression analysis using microarrays of synthetic long oligonucleotides is limited in that it requires substantial amounts of RNA. To obtain these quantities from minute amounts of starting material, protocols were developed that linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labelling by reverse transcription and hybridization to sense-oriented oligonucleotide arrays. Here, we introduce a novel protocol in which aRNA labelling is achieved by a combination of two reverse and one forward transcription reactions followed by dye-incorporation using Klenow fragment, generating fluorescent antisense cDNA. We demonstrate high fidelity in arrays using up to 10(5)-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples. These results demonstrate that our protocol describes an efficient and reliable technique to expand the applicability of oligonucleotide arrays to studies where RNA is the limited source material.

Ablation of gap junctional communication in hepatocytes of transgenic mice does not lead to disrupted cellular homeostasis or increased spontaneous tumourigenesis.

Gap junctions between murine hepatocytes are composed of two subunit proteins, connexin26 (Cx26) and connexin32 (Cx32). Previously, we found increased formation of chemically induced liver tumours but no increase in spontaneous development of preneoplastic hepatic foci in mice that lacked Cx32 and expressed decreased amounts of Cx26. In order to clarify this tumour-suppressive effect and to overcome embryonic lethality of constitutive Cx26-deficient mice, cell type-specific targeting of the Cx26 gene was performed. Mice with loxP-flanked Cx26 coding DNA were crossed with mice expressing the Cre recombinase exclusively in hepatocytes. Progeny mice lacking Cx26 in the liver were viable and fertile with no obvious signs of phenotypic alterations. To generate mice that totally lack gap junctional intercellular coupling, these mice were crossed with constitutive Cx32-deficient mice. We found no increase in spontaneously induced liver tumour formation in Cx26 and double deficient Cx26/Cx32 mice. Occasionally, double deficient livers exhibited morphological alterations, like amyloidosis, and a slightly increased basal proliferation rate of hepatocytes. Although the absence of gap junction channels led to altered expression of adhesion-related proteins like E-cadherin and actin, microarray analyses of total liver transcripts yielded only few differences between Cx26-deficient and double deficient livers compared to control samples. Our results suggest that total lack of gap junctional communication due to hepatocytic ablation of Cx26 and Cx32 does not drastically alter basal hepatocytic function and does not lead to increased spontaneous liver tumour formation.

Differential gene expression in periportal and perivenous mouse hepatocytes.

Hepatocytes located in the periportal and perivenous zones of the liver lobule show remarkable differences in the levels and activities of various enzymes and other proteins. To analyze global gene expression patterns of periportal and perivenous hepatocytes, enriched populations of the two cell types were isolated by combined collagenase/digitonin perfusion from mouse liver and used for microarray analysis. In total, 198 genes and expressed sequences were identified that demonstrated a >/= 2-fold difference in expression between hepatocytes from the two different zones of the liver. A subset of 20 genes was additionally analyzed by real-time RT-PCR, validating the results obtained by the microarray analysis. Several of the differentially expressed genes encoded key enzymes of intermediary metabolism, including those involved in glycolysis and gluconeogenesis, fatty acid degradation, cholesterol and bile acid metabolism, amino acid degradation and ammonia utilization. In addition, several enzymes of phase I and phase II of xenobiotic metabolism were differentially expressed in periportal and perivenous hepatocytes. Our results confirm previous findings on metabolic zonation in liver, and extend our knowledge of the regulatory mechanisms at the transcriptional level.

Contrast-enhanced ultrasound in dermatomyositis- and polymyositis.

OBJECTIVE: To evaluate prospectively contrast-enhanced ultrasound (CEUS) in patients suspected of having dermatomyositis or polymyositis. METHODS: In 35 patients (23 women, 12 men; mean age, 51 years+/-16 years) who were suspected of having dermatomyositis or polymyositis, perfusion in clinically affected skeletal muscles was quantified with contrast-enhanced intermittent power Doppler ultrasound. By applying a modified model that analyzed the replenishment kinetics of microbubbles, the perfusion-related parameters blood flow, local blood volume and blood flow velocity were measured. Findings were compared with muscle biopsy appearances and with the results of MRI that was performed with a 1.5-Tesla unit. Receiver operating characteristic analysis was performed and optimum thresholds for diagnosis of myositis were determined. RESULTS: Eleven patients had histologically confirmed dermatomyositis or polymyositis and showed significantly higher blood flow velocity (P=.01 for dermato- and P<.001 for polymyositis), blood flow (P<.001 for dermato- and polymyositis), and blood volume (P=.007 for dermato- and P<.001 for polymyositis) on contrast-enhanced ultrasound than those who did not have myositis. An increase in signal intensity on T2-weighted MR images was found in all patients with myositis. MRI had a sensitivity, specificity, positive (PPV), and negative predicting values (NPV) of 100%, 88%, 77%, and 100% for diagnosis of myositis, respectively. CEUS blood flow was the best ultrasound measure for diagnosis of dermato- or polymyositis with sensitivity, specificity, PPV, and NPV of 73%, 91%, 80%, and 88%, respectively. CONCLUSIONS: Increased skeletal muscle perfusion measured by CEUS could serve as an additional measurer for the diagnosis of an inflammatory myopathy.

A constitutively active dioxin/aryl hydrocarbon receptor promotes hepatocarcinogenesis in mice.

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding enzymes involved in drug metabolism. Known ligands include polycyclic aromatic hydrocarbons, certain polychlorinated biphenyls, and the polyhalogenated dioxins including 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both polyhalogenated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin are potent promoters of rodent hepatocarcinogenesis in two-stage initiation-promotion experiments. Although several lines of evidence indicate the involvement of the AhR in toxic effects mediated by polyhalogenated biphenyls and dioxins, its involvement in tumor promotion has not been unequivocally proven. In the present study, a transgenic mouse line expressing a constitutively active AhR (CA-AhR) has been used to investigate the role of the AhR in hepatocarcinogenesis. Male AhR wild-type and CA-AhR-transgenic B6C3F1-mice were treated with a single injection of the hepatocarcinogen N-nitrosodiethylamine at 6 weeks of age and were subsequently kept untreated on control diet. Thirty five weeks after carcinogen treatment, mice were sacrificed, and the prevalence and multiplicity of liver tumors were determined. Whereas only 1 small liver tumor was observed in 15 AhR-wild-type mice, 19 tumors (two >1 cm in diameter) were present in 18 CA-AhR-transgenic mice. This result demonstrates the oncogenic potential of the activated AhR and implicates an important role of the receptor in promotion of hepatocarcinogenesis. A microarray-based gene expression-profiling analysis revealed down-regulation in the liver of CA-AhR-transgenic mice of a cluster of genes encoding heat shock proteins, including GRP78/BiP, Herp1, Hsp90, DnaJ (Hsp40) homologue B1, and Hsp105, which are important for protein folding and quality control.

Influence of field potential duration on spontaneous beating rate of human induced pluripotent stem cell-derived cardiomyocytes: Implications for data analysis and test system selection.

INTRODUCTION: Field potential duration in human pluripotent stem cell (hiPSC)-derived cardiomyocytes is discussed as parameter for the assessment of drug-induced delayed repolarization. In spontaneously beating hiPSC-derived cardiomyocytes field potential duration varies depending on beating rate but beating rate can also be influenced by field potential duration. This interdependence is not fully understood and therefore mandates careful data analysis and cautious interpretation of the results. METHODS: We analysed data from several types of hiPSC-derived cardiomyocytes and, for comparison, primary embryonic chick cardiomyocytes using reference compounds to study the relationship between spontaneous rate and field potential duration. Based on such data we developed a method based on a regression model of drug-induced changes in the inter-beat interval versus changes in the field potential duration to distinguish primary rate from repolarisation effects. RESULTS: We demonstrate the application of this approach with reference and research compounds. Cells from different sources differed with regard to the direct or indirect effects of reference compounds on spontaneous beating. All cell types showed an adaptation of field potential duration upon rate changes induced by reference compounds, however, the adaptation of the spontaneous rate after compound-induced changes in field potential duration varied considerably between cell types. DISCUSSION: As shown by comparison with data from guinea pig papillary muscle, an ex vivo model with a fixed stimulation rate, this approach is more appropriate than the application of correction algorithms routinely used for in vivo data since such algorithms do not account for a dependence of rate on field potential duration.

RNAi Screen for NRF2 Inducers Identifies Targets That Rescue Primary Lung Epithelial Cells from Cigarette Smoke Induced Radical Stress.

Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism.