[When to suspect pulmonary embolism: a balancing act]. / Wanneer moet je aan een longembolie denken?

Pulmonary embolism (PE) is a common disease, which can present with a variety of symptoms. Optimal use of diagnostics is challenging given the tight and delicate balance between underdiagnosis and over-testing or overdiagnosis. Diagnostic delay occurs in a substantial part of patients, and seems more common in those with known cardiopulmonary disease or non-specific signs and symptoms. At the other end of the spectrum, the amount of diagnostic imaging increases. Increased use of diagnostic imaging in general leads to more harmful exposures and might result in overtreatment, as may be the case in subsegmental PE. Correct use of clinical prediction rules reduces the need for diagnostic imaging while PE can still be ruled out safely. This clinical lesson describes three cases of PE and provides an overview of factors that contribute to underdiagnosis or overdiagnosis. We provide recommendations to improve our balancing act for this challenging disease.

Tailored anticoagulant treatment after a first venous thromboembolism: protocol of the Leiden Thrombosis Recurrence Risk Prevention (L-TRRiP) study - cohort-based randomised controlled trial.

INTRODUCTION: Patients with a first venous thromboembolism (VTE) are at risk of recurrence. Recurrent VTE (rVTE) can be prevented by extended anticoagulant therapy, but this comes at the cost of an increased risk of bleeding. It is still uncertain whether patients with an intermediate recurrence risk or with a high recurrence and high bleeding risk will benefit from extended anticoagulant treatment, and whether a strategy where anticoagulant duration is tailored on the predicted risks of rVTE and bleeding can improve outcomes. The aim of the Leiden Thrombosis Recurrence Risk Prevention (L-TRRiP) study is to evaluate the outcomes of tailored duration of long-term anticoagulant treatment based on individualised assessment of rVTE and major bleeding risks. METHODS AND ANALYSIS: The L-TRRiP study is a multicentre, open-label, cohort-based, randomised controlled trial, including patients with a first VTE. We classify the risk of rVTE and major bleeding using the L-TRRiP and VTE-BLEED scores, respectively. After 3 months of anticoagulant therapy, patients with a low rVTE risk will discontinue anticoagulant treatment, patients with a high rVTE and low bleeding risk will continue anticoagulant treatment, whereas all other patients will be randomised to continue or discontinue anticoagulant treatment. All patients will be followed up for at least 2 years. Inclusion will continue until the randomised group consists of 608 patients; we estimate to include 1600 patients in total. The primary outcome is the combined incidence of rVTE and major bleeding in the randomised group after 2 years of follow-up. Secondary outcomes include the incidence of rVTE and major bleeding, functional outcomes, quality of life and cost-effectiveness in all patients. ETHICS AND DISSEMINATION: The protocol was approved by the Medical Research Ethics Committee Leiden-Den Haag-Delft. Results are expected in 2028 and will be disseminated through peer-reviewed journals and during (inter)national conferences. TRIAL REGISTRATION NUMBER: NCT06087952.

The idiosyncratic drug-induced gene expression changes in HepG2 cells.

The inflammatory stress has been associated with an increase in susceptibility to idiosyncratic drug-induced liver injury (DILI). However, the molecular mechanisms of this inflammation-associated idiosyncratic drug hepatotoxicity remain unknown. We exposed HepG2 cells with high and low doses of three idiosyncratic (I) and three non-idiosyncratic (N) compounds, in the presence (I+ and N+) or absence (I- and N-) of a cytokine mix for 6, 12 and 24 h. To investigate the genome-wide expression patterns, microarray was performed using the Agilent 4×44K Whole Human Genome chips. The data presented in this DIB include the expression of genes participating in the ceramide metabolism, ER stress, apoptosis and cell survival pathways. The functions of these genes were illustrated in our associated article (Jiang et al., 2017) [1]. Raw and normalized gene expression data are available through NCBI GEO (accession number GSE102006).

Omics-based identification of the combined effects of idiosyncratic drugs and inflammatory cytokines on the development of drug-induced liver injury.

The mechanisms of idiosyncratic drug-induced hepatotoxicity remain largely unclear. It has demonstrated that the drug idiosyncrasy is potentiated in the context of inflammation and intracellular ceramides may play a role in this process. To study the mechanisms, HepG2 cells were co-treated with high and low doses of three idiosyncratic (I) and three non-idiosyncratic (N) compounds, with (I+ and N+) or without (I- and N-) a cytokine mix. Microarray, lipidomics and flow cytometry were performed to investigate the genome-wide expression patterns, the intracellular ceramide levels and the induction of apoptosis. We found that all I+ treatments significantly influenced the immune response- and response to stimulus-associated gene ontology (GO) terms, but the induction of apoptotic pathways, which was confirmed by flow cytometry, only appeared to be induced after the high-dose treatment. The ceramide signaling-, ER stress-, NF-kB activation- and mitochondrial activity-related pathways were biologically involved in apoptosis induced by the high-dose I+. Additionally, genes participating in ceramide metabolism were significantly altered resulting in a measurable increase in ceramide levels. The increases in ceramide concentrations may induce ER stress and activate the JNK pathway by affecting the expression of the related genes, and eventually trigger the mitochondria-independent apoptosis in hepatocytes. Overall, our study provides a potential mechanism to explain the role of inflammation in idiosyncratic drug reactions.

Learning to (dis)like: The effect of evaluative conditioning with tastes and faces on odor valence assessed by implicit and explicit measurements.

Evaluative conditioning may be an important mechanism for learning food preferences and aversions; however, in both real life and experimental settings it has not been consistently successful. The current study aimed to gain more insight into which underlying factors may contribute to a successful outcome of olfactory evaluative conditioning. Two groups of 18 participants came in on three consecutive days, and were repeatedly exposed to four novel, neutral odors (CS) coupled to varying disliked, neutral, liked, or no stimuli (taste and/or pictures, US), following a 50% reinforcement schedule, leading to 40 odor presentations per session. Liking ratings, as well as changes in the autonomic nervous system were assessed before, during and after conditioning. We were able to induce negative, but not positive, affective changes by pairing neutral odors with tastes and pictures differing in valence. Negative as well as multimodal stimuli appear to be more potent US, since they may be considered more salient. Lastly, results of the current study imply that heart rate is responsive to changes in valence of olfactory stimuli, and perhaps even more sensitive than explicit ratings of liking.

Pathways for ligand activated nuclear receptors to unravel the genomic responses induced by hepatotoxicants.

The liver is a vital organ in vertebrates that can be subject to disease, among others due to exposure to toxic xenobiotic compounds. A group of transcription factors named ligand activated nuclear receptors (LANR) influence and regulate important liver functions, and can be activated by many xenobiotic compounds, which thereby can cause hepatotoxicity. Systematic analysis of the gene pathways regulated by LANR using modern 'omics technologies is important for investigating modes-of-action of hepatotoxicants. So far, these pathways are not publicly available in a format that allows these studies. We used PathVisio to build liver-specific LANR pathways, both for rats and humans. Since many LANR pathways are linked to each other, we also merged them into a meta-pathway. The pathways are in a GPML-format that enables pathway statistics and visualisations, and will be made available to the public through WikiPathways. We demonstrate the performance of these novel pathways in evaluating transcriptomic studies from the Japanese toxicogenomics project database (Open TG-GATEs). We show that the new pathways can be used to accurately analyse and visualize the effects of prototypical hepatotoxicants in important liver processes, and thus to evaluate the possible mode-of-actions of hepatotoxic xenobiotic compounds by assessing which LANRs are possible targets.

Baseline and genotoxic compound induced gene expression profiles in HepG2 and HepaRG compared to primary human hepatocytes.

Efforts are put into developing toxicogenomics-based toxicity testing methods using in vitro human cell models for improving human risk assessment/replacing animal models. Human in vitro liver models include HepG2, HepaRG and primary human hepatocytes (PHH). Studies on comparability/applicability of these cell types mainly focus on assessing baseline biotransformation capacities/cytochrome P450-inducibility, but compound-induced gene expression profiles are at least as important. Therefore, we compared baseline and aflatoxin B1- and benzo(α)pyrene-induced gene expression profiles in HepG2, HepaRG and PHH (11-13 donors). At baseline, all liver models differ from each other with respect to whole genome gene expression levels. PHH show profound inter-individual differences, and are most similar to HepaRG. After compound exposure, induced gene expression profiles are more similar between cell models, especially for benzo(α)pyrene. Pathways involved in compound metabolism are induced in all 3 models, while others are more pronounced in a specific cell model. Examples are transcriptomic modifications of carbohydrate-related genes (HepaRG) and of receptor-related genes (PHH) after benzo(α)pyrene exposure, and of cell cycle-related genes (HepG2) after aflatoxin B1 exposure. PHH gene expression responses are the most heterogeneous. In conclusion, at base line level PHH are more similar to HepaRG than to HepG2, but for toxicogenomics applications both cell lines perform equally well in comparison to PHH.

Toxicogenomic profiles in relation to maternal immunotoxic exposure and immune functionality in newborns.

A crucial period for the development of the immune system occurs in utero. This results in a high fetal vulnerability to immunotoxic exposure, and indeed, immunotoxic effects have been reported, demonstrating negative effects on immune-related health outcomes and immune functionality. Within the NewGeneris cohort BraMat, a subcohort of the Norwegian Mother and Child Cohort Study (MoBa), immunotoxicity was demonstrated for polychlorinated biphenyls and dioxins, showing associations between estimated maternal intake levels and reduced measles vaccination responses in the offspring at the age of 3. The present study aimed to investigate this link at the transcriptomic level within the same BraMat cohort. To this end, whole-genome gene expression in cord blood was investigated and found to be associated with maternal Food Frequency Questionnaires-derived exposure estimates and with vaccination responses in children at 3 years of age. Because the literature reports gender specificity in the innate, humoral, and cell-mediated responses to viral vaccines, separate analysis for males and females was conducted. Separate gene sets for male and female neonates were identified, comprising genes significantly correlating with both 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and polychlorinated biphenyls (PCB) exposure and with measles vaccination response. Noteworthy, genes correlating negatively with exposure in general show positive correlations with antibody levels and vice versa. For both sexes, these included immune-related genes, suggesting immunosuppressive effects of maternal exposure to TCDD and PCB at the transcriptomic level in neonates in relation to measles vaccination response 3 years later.

A transcriptomics-based in vitro assay for predicting chemical genotoxicity in vivo.

The lack of accurate in vitro assays for predicting in vivo toxicity of chemicals together with new legislations demanding replacement and reduction of animal testing has triggered the development of alternative methods. This study aimed at developing a transcriptomics-based in vitro prediction assay for in vivo genotoxicity. Transcriptomics changes induced in the human liver cell line HepG2 by 34 compounds after treatment for 12, 24, and 48 h were used for the selection of gene-sets that are capable of discriminating between in vivo genotoxins (GTX) and in vivo nongenotoxins (NGTX). By combining transcriptomics with publicly available results for these chemicals from standard in vitro genotoxicity studies, we developed several prediction models. These models were validated by using an additional set of 28 chemicals. The best prediction was achieved after stratification of chemicals according to results from the Ames bacterial gene mutation assay prior to transcriptomics evaluation after 24h of treatment. A total of 33 genes were selected for discriminating GTX from NGTX for Ames-positive chemicals and 22 for Ames-negative chemicals. Overall, this method resulted in 89% accuracy and 91% specificity, thereby clearly outperforming the standard in vitro test battery. Transcription factor network analysis revealed HNF3a, HNF4a, HNF6, androgen receptor, and SP1 as main factors regulating the expression of classifiers for Ames-positive chemicals. Thus, the classical bacterial gene mutation assay in combination with in vitro transcriptomics in HepG2 is proposed as an upgraded in vitro approach for predicting in vivo genotoxicity of chemicals holding a great promise for reducing animal experimentations on genotoxicity.

Transcriptomic fingerprints in human peripheral blood mononuclear cells indicative of genotoxic and non-genotoxic carcinogenic exposure.

For evaluating genotoxic exposure in human populations a number of biomarkers has been successfully applied over the last 30 years to determine early biological effects due to exposure to carcinogens. Despite their success, these early biological effect markers provide limited mechanistic insight, and do not allow detection of exposure to non-genotoxic carcinogens. Gene expression profiling forms a promising tool for the development of new biomarkers in blood cells to overcome these limitations. The aim of our research was to identify novel genomics-based candidate markers for genotoxic and non-genotoxic carcinogen exposure in human peripheral blood cells (PBMC). Whole genome gene expression changes were investigated following 20 h of in vitro exposure to a high and low concentration of eight genotoxic and three non-genotoxic carcinogenic compounds using whole genome microarrays. Per condition, PBMC of five independent donors were exposed, all in the presence of human liver S9. Sets of genes, as well as biological pathways indicative of genotoxic exposure and of non-genotoxic carcinogenic exposure were identified. Furthermore, networks were built using the genotoxic and non-genotoxic gene sets, showing the majority of the genes to be interlinked and revealing distinctive transcription factors for both classes. The identification of these potential candidate marker genes might contribute to the development of genomic based biomarkers of carcinogen exposure.

Comparison of phenotypic and transcriptomic effects of false-positive genotoxins, true genotoxins and non-genotoxins using HepG2 cells.

The conventional in vitro assays for genotoxicity assessment of chemicals are characterised by a high false-positive rate, thus failing to correctly predict their in vivo genotoxic effects. This study aimed to identify the cellular mechanisms induced by the false-positive genotoxins quercetin, 8-Hydroxyquinoline and 17-beta oestradiol in comparison to true genotoxins and non-genotoxins, by combining in vitro phenotypic parameters with transcriptomics data from HepG2 cells. The effects of these compounds on the phosphorylation of H2AX, cell cycle distribution and whole genome gene expression following treatment for 12, 24 and 48 h were compared with the effects of true genotoxins [benzo[a]pyrene and aflatoxin B1] and non-genotoxins (2,3,7,8-tetrachlorodibenzodioxin, cyclosporin A and ampicillin C). Quercetin induced similar phenotypic effects as true genotoxins and to some extent similar gene expression alterations. Different gene expression changes were also observed, including the up-regulation of DNA repair-related genes. 8-Hydroxyquinoline and 17-beta oestradiol showed no similarities to the true genotoxins at both the phenotypic and the transcriptomic level. In a classification approach, classifiers were selected to discriminate between genotoxins and non-genotoxins. Subsequent analysis for the false-positive compounds showed quercetin to be predicted as genotoxic and 8-hydroxyquinoline and 17-beta oestradiol as non-genotoxic. Our results support that transcriptomics analysis of compound effects in HepG2 leads to similar results with phenotypic analysis and provides additional mechanistic information. Therefore, combined evaluation of gene expression alterations and relevant functional end points using HepG2 cells may contribute to the better understanding of modes-of-action of chemicals and the correct evaluation of their genotoxic properties.

In vitro cytokine release from human peripheral blood mononuclear cells in the assessment of the immunotoxic potential of chemicals.

Alternative methods to the use of animals in testing of chemicals are needed. We investigated if the immunotoxic potential of 12 dietary toxicants could be predicted from effects on cytokine release from human peripheral blood mononuclear cells (PBMC) after in vitro exposure. Nine cytokines were selected to reflect different types of immune responses. The toxicants were classified as immunotoxic or non-immunotoxic substances according to the published in vivo data. Isolated human PBMC were exposed for 20 h to three concentrations of each of the 12 substances in the presence of human liver S9 fraction. After further incubation of PBMC in fresh medium containing the mitogen phytohemagglutinin (PHA, 10 µg/ml) for 48 h, release of the nine selected cytokines into the supernatant as well as cell proliferation were measured by Luminex technology™ and the BrdU incorporation assay, respectively. All 12 substances investigated affected the release of one or more cytokines, and each of the substances showed different cytokine release patterns. Within the limitations of the study design, the present study suggests that the effect of the substances on mitogen-induced cytokine release from PBMC cannot predict their immunotoxic potential, but may be useful in mechanistic studies.

Gene expression profiling in primary mouse hepatocytes discriminates true from false-positive genotoxic compounds.

Well-established in vitro methods for testing the genotoxic potency of chemicals--such as the Ames/Salmonella test, the mouse lymphoma assay, the micronucleus test and the chromosomal aberration test--show a high false-positive rate for predicting in vivo genotoxicity and carcinogenicity. Thus, there is a need for more reliable in vitro assays. We investigated whether gene expression profiling in metabolically competent primary mouse hepatocytes is capable of discriminating true genotoxic (GTX) compounds from false-positive genotoxic (FP-GTX) compounds. Sandwich-cultured primary hepatocytes from male C57Bl6 mice were treated for 24 and 48 h with five true GTX and five FP-GTX compounds. Whole genome gene expression modifications were analysed by means of Affymetrix mouse genome 430 2.0 microarrays. Filtered genes were used for hierarchical clustering and class prediction methods. Classifiers were generated by prediction analysis of microarray using a leave-one-compound-out method and selecting the genes that were common to the 10 training sets. For the training compounds, all but one were correctly classified. Validation of the classification model with five new compounds resulted in a 100% correct classification at 24 h and 80% at 48 h. The generated classifiers were mostly involved in metabolic and biosynthetic processes, immune responses and apoptosis. Applying genes whose expression change correlates with γH2AX foci, a measure for DNA damage, did not improve the classification. The present study shows that gene expression profiling in primary mouse hepatocytes is capable of discriminating between true GTX and FP-GTX compounds.

A pro-inflammatory genotype predisposes to Barrett's esophagus.

INTRODUCTION: Severity of mucosal inflammation is shown to be associated with Barrett's esophagus (BE) development in animals. It has therefore been postulated that a strong pro-inflammatory host response predisposes to BE. AIM: To determine the impact of cytokine gene polymorphisms on the development of BE. METHODS: The multiplex SNaPshot method was used to determine interleukin (IL)-12B (A+1188C), IL-10 (C-592A, C-819T, A-1082G), IL-8 (A-251T), IL-6 (G-174C) and IL-2 (G-330T) gene polymorphisms in 255 patients with BE and 247 patients with reflux esophagitis (RE). RESULTS: The presence of the IL-12B C-allele, which is associated with increased IL-12p70 expression, was more frequently observed in BE than in RE patients [odds ratio (OR) 1.8; 95% confidence interval (CI) 1.2-2.7; P = 0.007). The risk of BE was increased in patients in whom the IL-12B C-allele coincided with a hiatal hernia (OR 2.9; 95% CI 1.32-6.58; P = 0.008). The IL-10(-1082) GG genotype, which is associated with higher IL-10 levels, was also associated with a decreased risk of BE when it was associated with the IL-12B C-allele, indicating IL-10-dependent down-regulation of IL-12p70 expression. A combination of the IL-12B AA genotype and the IL-10 AA or AG genotypes was associated with RE (OR 1.4; 95% CI 1.05-1.85; P = 0.011). CONCLUSION: A genetic profile predisposing to a strong pro-inflammatory host response, mediated by IL-12p70 and partially dependent on IL-10, is associated with BE. This risk further increases when this genotype coincides with a hiatal hernia, suggesting that exposure to gastroesophageal reflux in the presence of a pro-inflammatory genetic background is a driving force in the development of BE.

Dose reduction in digital chest radiography and perceived image quality.

The aim of the study was to investigate whether radiologists can rank the image quality of digital radiographs with different doses; a preliminary study investigated whether reduced dose images provide sufficient diagnostic quality. Raw data of 40 chest radiographs (posteroanterior (PA) and lateral) obtained with a full-field slot-scan charge-coupled device system in 20 patients with chest pathology were used. Noise was added to simulate reduced dose levels to 50%, 25% and 12%. Four observers ranked the quality of the corresponding images and judged the diagnostic quality. Linear regression analysis was performed. Differences were found in image quality at the different dose levels for both PA (p

The importance of polymerization and galloylation for the antiproliferative properties of procyanidin-rich natural extracts.

Grape (Vitis vinifera) and pine (Pinus pinaster) bark extracts are widely used as nutritional supplements. Procyanidin-rich fractions from grape and pine bark extract showing different mean degrees of polymerization, percentage of galloylation (percentage of gallate esters) and reactive oxygen species-scavenging capacity were tested on HT29 human colon cancer cells. We observed that the most efficient fractions in inhibiting cell proliferation, arresting the cell cycle in G(2) phase and inducing apoptosis were the grape fractions with the highest percentage of galloylation and mean degree of polymerization. Additionally, the antiproliferative effects of grape fractions were consistent with their oxygen radical-scavenging capacity and their ability to trigger DNA condensation-fragmentation.

A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling.

A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and beta-naphthoflavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modified model was evaluated by comparing its genome-wide expression profiles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modified model resembled more closely the in vivo situation. Gene expression results revealed large differences between in vivo and both in vitro models. The slight differences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modified model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities corresponded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modified model may be preferred.

Genome-wide differential gene expression in children exposed to air pollution in the Czech Republic.

The Teplice area in the Czech Republic is a mining district where elevated levels of air pollution including airborne carcinogens, have been demonstrated, especially during winter time. This environmental exposure can impact human health; in particular children may be more vulnerable. To study the impact of air pollution in children at the transcriptional level, peripheral blood cells were subjected to whole genome response analysis, in order to identify significantly modulated biological pathways and processes as a result of exposure. Using genome-wide oligonucleotide microarrays, we investigated differential gene expression in children from the Teplice area (n=23) and compared them with children from the rural control area of Prachatice (n=24). In an additional approach, individual gene expressions were correlated with individual peripheral blood lymphocyte micronuclei frequencies, in order to evaluate the linkage of individual gene expressions with an established biomarker of effect that is representative for increased genotoxic risk. Children from the Teplice area showed a significantly higher average micronuclei frequency than Prachatice children (p=0.023). For considerable numbers of genes, the expression differed significantly between the children from the two areas. Amongst these genes, considerable numbers of genes were observed to correlate significantly with the frequencies of micronuclei. The main biological process that appeared significantly affected overall was nucleosome assembly. This suggests an effect of air pollution on the primary structural unit of the condensed DNA. In addition, several other pathways were modulated. Based on the results of this study, we suggest that transcriptomic analysis represents a promising biomarker for environmental carcinogenesis.

Comparison of supervised clustering methods to discriminate genotoxic from non-genotoxic carcinogens by gene expression profiling.

Prediction of the toxic properties of chemicals based on modulation of gene expression profiles in exposed cells or animals is one of the major applications of toxicogenomics. Previously, we demonstrated that by Pearson correlation analysis of gene expression profiles from treated HepG2 cells it is possible to correctly discriminate and predict genotoxic from non-genotoxic carcinogens. Since to date many different supervised clustering methods for discrimination and prediction tests are available, we investigated whether application of the methods provided by the Whitehead Institute and Stanford University improved our initial prediction. Four different supervised clustering methods were applied for this comparison, namely Pearson correlation analysis (Pearson), nearest shrunken centroids analysis (NSC), K-nearest neighbour analysis (KNN) and Weighted voting (WV). For each supervised clustering method, three different approaches were followed: (1) using all the data points for all treatments, (2) exclusion of the samples with marginally affected gene expression profiles and (3) filtering out the gene expression signals that were hardly altered. On the complete data set, NSC, KNN and WV outperformed the Pearson test, but on the reduced data sets no clear difference was observed. Exclusion of samples with marginally affected profiles improved the prediction by all methods. For the various prediction models, gene sets of different compositions were selected; in these 27 genes appeared three times or more. These 27 genes are involved in many different biological processes and molecular functions, such as apoptosis, cell cycle control, regulation of transcription, and transporter activity, many of them related to the carcinogenic process. One gene, BAX, was selected in all 10 models, while ZFP36 was selected in 9, and AHR, MT1E and TTR in 8. Summarising, this study demonstrates that several supervised clustering methods can be used to discriminate certain genotoxic from non-genotoxic carcinogens by gene expression profiling in vitro in HepG2 cells. None of the methods clearly outperforms the others.