Costs of molecular adaptation to the chemical exposome: a focus on xenobiotic metabolism pathways.

Organisms adapt to their environment through different pathways. In vertebrates, xenobiotics are detected, metabolized and eliminated through the inducible xenobiotic-metabolizing pathways (XMP) which can also generate reactive toxic intermediates. In this review, we will discuss the impacts of the chemical exposome complexity on the balance between detoxication and side effects. There is a large discrepancy between the limited number of proteins involved in these pathways (few dozens) and the diversity and complexity of the chemical exposome (tens of thousands of chemicals). Several XMP proteins have a low specificity which allows them to bind and/or metabolize a large number of chemicals. This leads to undesired consequences, such as cross-inhibition, inefficient metabolism, release of toxic intermediates, etc. Furthermore, several XMP proteins have endogenous functions that may be disrupted upon exposure to exogenous chemicals. The gut microbiome produces a very large number of metabolites that enter the body and are part of the chemical exposome. It can metabolize xenobiotics and either eliminate them or lead to toxic derivatives. The complex interactions between chemicals of different origins will be illustrated by the diverse roles of the aryl hydrocarbon receptor which binds and transduces the signals of a large number of xenobiotics, microbiome metabolites, dietary chemicals and endogenous compounds. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

Cigarette smoke and tumor microenvironment copromote aggressiveness of human breast cancer cells.

Breast cancer is a major public health issue and the role of pollutants in promoting breast cancer progression has recently been suggested. We aimed to assess if a mixture of pollutants, cigarette smoke, could favor the aggressivity of breast cancer cells. We also evaluated the impact of the tumor microenvironment, largely represented by adipocytes, in mediating this modification of cell phenotype. Breast cancer cells lines, MCF-7 were cultured using a transwell coculture model with preadipocytes hMADS cells or were cultured alone. Cells were treated with cigarette smoke extract (CSE) and the four conditions: control, treated by CSE, coculture, and coexposure (coculture and CSE) were compared. We analyzed morphological changes, cell migration, resistance to anoikis, stemness, epithelial-to-mesenchymal transition (EMT), and the presence of hormonal receptors in each condition. A complete transcriptomic analysis was carried out to highlight certain pathways. We also assessed whether the aryl hydrocarbon receptor (AhR), a receptor involved in the metabolism of xenobiotics, could mediate these modifications. Several hallmarks of metastasis were specific to the coexposure condition (cell migration, resistance to anoikis, stemness characterized by CD24/CD44 ratios and ALDH1A1 and ALDH1A3 rates) whereas others (morphological changes, EMT, loss of hormonal receptors) could be seen in the coculture condition and were aggravated by CSE (coexposure). Moreover, MCF-7 cells presented a decrease in hormonal receptors, suggesting an endocrine treatment resistance. These results were confirmed by the transcriptomic analysis. We suggest that the AhR could mediate the loss of hormonal receptor and the increase in cell migration.

Impact of mixtures of persistent organic pollutants on breast cancer aggressiveness.

INTRODUCTION: Breast cancer (BC) is frequent with a poor prognosis in case of metastasis. The role of the environment has been poorly evaluated in its progression. We searched to assess whether a mixture of pollutants could be responsible of BC aggressiveness. METHODS: Patients undergoing surgery for their BC were prospectively included in the METAPOP cohort. Forty-two POPs were extracted, among them 17 dioxins (PCDD/F), 16 polychlorobiphenyls (PCB), 8 polybromodiphenylethers (PBDE) and 2,2',4,4',5,5'-hexabromobiphenyl (PBB153) were measured in the adipose tissue surrounding the tumor. BC aggressiveness was defined using tumor size and metastasis (distant or lymph nodes). Two complementary models were used to evaluate the impact of the mixture of pollutants: the BKMR (Bayesian Kernel machine regression) and WQS (weighted quantile sum regression) models. The WQS estimates the weight (positive or negative) of a certain chemical based on its quantile and the BKMR model applies a kernel-based approach to estimate posterior inclusion probabilities. The sub-group of patients with a body mass index (BMI) > 22 kg/ m2 was also analyzed. RESULTS: Ninety-one patients were included. Of these, 38 patients presented a metastasis, and the mean tumor size was 25.4 mm. The mean BMI was 24.5 kg/m2 (+/- 4.1). No statistical association was found in the general population. However, in patients with a BMI > 22 kg/ m2, our mixture was positively associated with tumor size (OR: 9.73 95 %CI: 1.30-18.15) and metastasis (OR = 3.98 95 %CI = 1.09-17.53) using the WQS model. Moreover, using the BKMR model on chemical families, dioxin like chemicals and PCDD were associated with a higher risk of metastasis. DISCUSSION: These novel findings identified a mixture associated with breast cancer aggressiveness in patients with a BMI > 22 kg/ m2.

Adverse outcome pathway from activation of the AhR to breast cancer-related death.

Adverse outcome pathways (AOPs) are formalized and structured linear concepts that connect one molecular initiating event (MIE) to an adverse outcome (AO) via different key events (KE) through key event relationships (KER). They are mainly used in eco-toxicology toxicology, and regulatory health issues. AOPs must respond to specific guidelines from the Organization for Economic Co-operation and Development (OECD) to weight the evidence between each KE. Breast cancer is the deadliest cancer in women with a poor prognosis in case of metastatic breast cancer. The role of the environments in the formation of metastasis has been suggested. We hypothesized that activation of the AhR (MIE), a xenobiotic receptor, could lead to breast cancer related death (AO), through different KEs, constituting a new AOP. An artificial intelligence tool (AOP-helpfinder), which screens the available literature, was used to collect all existing scientific abstracts to build a novel AOP, using a list of key words. Four hundred and seven abstracts were found containing at least a word from our MIE list and either one word from our AO or KE list. A manual curation retained 113 pertinent articles, which were also screened using PubTator. From these analyses, an AOP was created linking the activation of the AhR to breast cancer related death through decreased apoptosis, inflammation, endothelial cell migration, angiogenesis, and invasion. These KEs promote an increased tumor growth, angiogenesis and migration which leads to breast cancer metastasis and breast cancer related death. The evidence of the proposed AOP was weighted using the tailored Bradford Hill criteria and the OECD guidelines. The confidence in our AOP was considered strong. An in vitro validation must be carried out, but our review proposes a strong relationship between AhR activation and breast cancer-related death with an innovative use of an artificial intelligence literature search.

Identification of Modulators of the C. elegans Aryl Hydrocarbon Receptor and Characterization of Transcriptomic and Metabolic AhR-1 Profiles.

The Aryl hydrocarbon Receptor (AhR) is a xenobiotic sensor in vertebrates, regulating the metabolism of its own ligands. However, no ligand has been identified to date for any AhR in invertebrates. In C. elegans, the AhR ortholog, AHR-1, displays physiological functions. Therefore, we compared the transcriptomic and metabolic profiles of worms expressing AHR-1 or not and investigated the putative panel of chemical AHR-1 modulators. The metabolomic profiling indicated a role for AHR-1 in amino acids, carbohydrates, and fatty acids metabolism. The transcriptional profiling in neurons expressing AHR-1, identified 95 down-regulated genes and 76 up-regulated genes associated with neuronal and metabolic functions in the nervous system. A gene reporter system allowed us to identify several AHR-1 modulators including bacterial, dietary, or environmental compounds. These results shed new light on the biological functions of AHR-1 in C. elegans and perspectives on the evolution of the AhR functions across species.

Aryl Hydrocarbon Receptor and Its Diverse Ligands and Functions: An Exposome Receptor.

The aryl hydrocarbon receptor (AhR) is a transcriptional factor that regulates multiple functions following its activation by a variety of ligands, including xenobiotics, natural products, microbiome metabolites, and endogenous molecules. Because of this diversity, the AhR constitutes an exposome receptor. One of its main functions is to regulate several lines of defense against chemical insults and bacterial infections. Indeed, in addition to its well-established detoxication function, it has several functions at physiological barriers, and it plays a critical role in immunomodulation. The AhR is also involved in the development of several organs and their homeostatic maintenance. Its activity depends on the type of ligand and on the time frame of the receptor activation, which can be either sustained or transient, leading in some cases to opposite modes of regulations as illustrated in the regulation of different cancer pathways. The development of selective modulators and their pharmacological characterization are important areas of research.

Identification of non-validated endocrine disrupting chemical characterization methods by screening of the literature using artificial intelligence and by database exploration.

BACKGROUND: Exposure to endocrine disrupting chemicals (EDCs) represents a critical public health threat. Several adverse health outcomes (e.g., cancers, metabolic and neurocognitive/neurodevelopmental disorders, infertility, immune diseases and allergies) are associated with exposure to EDCs. However, the regulatory tests that are currently employed in the EU to identify EDCs do not assess all of the endocrine pathways. OBJECTIVE: Our objective was to explore the literature, guidelines and databases to identify relevant and reliable test methods which could be used for prioritization and regulatory pre-validation of EDCs in missing and urgent key areas. METHODS: Abstracts of articles referenced in PubMed were automatically screened using an updated version of the AOP-helpFinder text mining approach. Other available sources were manually explored. Exclusion criteria (computational methods, specific tests for estrogen receptors, tests under validation or already validated, methods accepted by regulatory bodies) were applied according to the priorities of the French Public-privatE Platform for the Pre-validation of Endocrine disRuptors (PEPPER) characterisation methods. RESULTS: 226 unique non-validated methods were identified. These experimental methods (in vitro and in vivo) were developed for 30 species using diverse techniques (e.g., reporter gene assays and radioimmunoassays). We retrieved bioassays mainly for the reproductive system, growth/developmental systems, lipogenesis/adipogenicity, thyroid, steroidogenesis, liver metabolism-mediated toxicity, and more specifically for the androgen-, thyroid hormone-, glucocorticoid- and aryl hydrocarbon receptors. CONCLUSION: We identified methods to characterize EDCs which could be relevant for regulatory pre-validation and, ultimately for the efficient prevention of EDC-related severe health outcomes. This integrative approach highlights a successful and complementary strategy which combines computational and manual curation approaches.

Aggressiveness and Metastatic Potential of Breast Cancer Cells Co-Cultured with Preadipocytes and Exposed to an Environmental Pollutant Dioxin: An in Vitro and in Vivo Zebrafish Study.

BACKGROUND: Breast cancer (BC) is a major public health concern, and its prognosis is very poor once metastasis occurs. The tumor microenvironment and chemical pollution have been suggested recently to contribute, independently, to the development of metastatic cells. The BC microenvironment consists, in part, of adipocytes and preadipocytes in which persistent organic pollutants (POPs) can be stored. OBJECTIVES: We aimed to test the hypothesis that these two factors (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an extensively studied, toxic POP and the microenvironment) may interact to increase tumor aggressiveness. METHODS: We used a co-culture model using BC MCF-7 cells or MDA-MB-231 cells together with hMADS preadipocytes to investigate the contribution of the microenvironment and 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD on BC cells. Global differences were characterized using a high-throughput proteomic assay. Subsequently we measured the BC stem cell-like activity, analyzed the cell morphology, and used a zebrafish larvae model to study the metastatic potential of the BC cells. RESULTS: We found that coexposure to TCDD and preadipocytes modified BC cell properties; moreover, it induced the expression of ALDH1A3, a cancer stem cell marker, and the appearance of giant cancer cells with cell-in-cell structures (CICs), which are associated with malignant metastatic progression, that we demonstrated in vivo. DISCUSSION: The results of our study using BC cell lines co-cultured with preadipocytes and a POP and an in vivo zebrafish model of metastasis suggest that the interactions between BC cells and their microenvironment could affect their invasive or metastatic potential. https://doi.org/10.1289/EHP7102.

Mitochondrial Dysfunction as a Hallmark of Environmental Injury.

Environmental factors including diet, sedentary lifestyle and exposure to pollutants largely influence human health throughout life. Cellular and molecular events triggered by an exposure to environmental pollutants are extremely variable and depend on the age, the chronicity and the doses of exposure. Only a fraction of all relevant mechanisms involved in the onset and progression of pathologies in response to toxicants has probably been identified. Mitochondria are central hubs of metabolic and cell signaling responsible for a large variety of biochemical processes, including oxidative stress, metabolite production, energy transduction, hormone synthesis, and apoptosis. Growing evidence highlights mitochondrial dysfunction as a major hallmark of environmental insults. Here, we present mitochondria as crucial organelles for healthy metabolic homeostasis and whose dysfunction induces critical adverse effects. Then, we review the multiple mechanisms of action of pollutants causing mitochondrial toxicity in link with chronic diseases. We propose the Aryl hydrocarbon Receptor (AhR) as a model of "exposome receptor", whose activation by environmental pollutants leads to various toxic events through mitochondrial dysfunction. Finally, we provide some remarks related to mitotoxicity and risk assessment.

Environmental chemicals, breast cancer progression and drug resistance.

Breast cancer (BC) is one of the most common causes of cancer in the world and the second leading cause of cancer deaths among women. Mortality is associated mainly with the development of metastases. Identification of the mechanisms involved in metastasis formation is, therefore, a major public health issue. Among the proposed risk factors, chemical environment and pollution are increasingly suggested to have an effect on the signaling pathways involved in metastatic tumor cells emergence and progression. The purpose of this article is to summarize current knowledge about the role of environmental chemicals in breast cancer progression, metastasis formation and resistance to chemotherapy. Through a scoping review, we highlight the effects of a wide variety of environmental toxicants, including persistent organic pollutants and endocrine disruptors, on invasion mechanisms and metastatic processes in BC. We identified the epithelial-to-mesenchymal transition and cancer-stemness (the stem cell-like phenotype in tumors), two mechanisms suspected of playing key roles in the development of metastases and linked to chemoresistance, as potential targets of contaminants. We discuss then the recently described pro-migratory and pro-invasive Ah receptor signaling pathway and conclude that his role in BC progression is still controversial. In conclusion, although several pertinent pathways for the effects of xenobiotics have been identified, the mechanisms of actions for multiple other molecules remain to be established. The integral role of xenobiotics in the exposome in BC needs to be further explored through additional relevant epidemiological studies that can be extended to molecular mechanisms.

First evidence of aryl hydrocarbon receptor as a druggable target in hypertension induced by chronic intermittent hypoxia.

BACKGROUND AND PURPOSE: Obstructive sleep apnea (OSA) is associated to a high prevalence of resistant arterial hypertension (HTN) justifying the research on novel targets. Chronic intermittent hypoxia (CIH) is a key feature in the development of OSA comorbidities, including HTN. EXPERIMENTAL APPROACH: We used a rat model of CIH-induced HTN to disclose the hypothesis that the aryl hydrocarbon receptor (AHR) is activated by CIH once it shares the same binding partner of HIF-1α and promotes pro-oxidant, pro-inflammatory (NF-kB) and pro-fibrotic events in common with CIH. KEY RESULTS: Upon established hypertension (21 days exposure to CIH), we observed an increase in Cyp1a1 mRNA in kidney cortex (6-fold), kidney medulla (3-fold) and liver (3-fold), but not in other tissues. Increased renal expression of Ahr and markers of inflammation (Rela), epithelial to mesenchymal transition markers, the rate-controlling step of gluconeogenesis, Pepck1, and members of HIF-pathway, namely, Hif3a were also observed. Daily administration (14 days) of AHR antagonist, CH-223191 (5 mg.kg-1.day-1, gavage), simultaneously to CIH prevented the increase in systolic blood pressure (SBP) by 53 ± 12% and in diastolic blood pressure (DBP) by 44 ± 16%. Moreover, its administration (14 days) upon already established HTN reversed the increase in SBP by 52 ± 12%. CONCLUSION AND IMPLICATIONS: CIH caused an activation of AHR signaling particularly in the kidney and its pharmacological blockade had a significant impact reverting already established HTN. This first evidence inspires innovative research opportunities for the understanding and treatment of this particular type of HTN.

Associations between persistent organic pollutants and risk of breast cancer metastasis.

BACKGROUND: Breast cancer (BC) is a major public health concern with over 2 million new cases diagnosed and over 600,000 deaths in 2018 in women worldwide. When distant metastases are present at diagnosis, the 5-year survival rate is only 26%. Recent studies have suggested that persistent organic pollutants (POPs) that accumulate in adipose tissue (AT) can influence tumor phenotype and stimulate cellular processes important for metastasis such as invasion. We, therefore, tested the hypothesis that POP exposure is associated with BC metastasis. METHODS: We conducted an exploratory case-control study in which the concentrations of 49 POPs were measured in both AT and serum samples from BC patients, with or without lymph node metastasis, who underwent partial or total mastectomies, lymph node biopsies and sampling of the adipocytic tumor microenvironment. Adjusted, unconditional logistic models were used to study the associations between the POP concentrations and the risk of metastasis and other hallmarks of cancer aggressiveness. RESULTS: 2.3.7.8-TCDD concentrations in AT are positively associated with the risk of metastasis in 43 patients who have BMIs equal or higher than 25 kg/m2 (odds ratio: 4.48 (1.32-20.71)). Furthermore, the concentrations of 2.3.7.8-TCDD and two coplanar PCBs (77&169) in AT also were positively associated with the risk of lymph node metastasis and the tumor size. CONCLUSION: Our study suggests that 2.3.7.8-TCDD and some PCBs contribute to the development of tumor metastasis and other hallmarks of cancer aggressiveness. While these results should be considered with caution, this is the first study to identify such potential risk factors. Larger longitudinal studies are necessary to confirm our results. Clinical Trial Protocol Record: 2013-A00663-42.

The Cellular Prion Protein Controls Notch Signaling in Neural Stem/Progenitor Cells.

The prion protein is infamous for its involvement in a group of neurodegenerative diseases known as Transmissible Spongiform Encephalopathies. In the longstanding quest to decipher the physiological function of its cellular isoform, PrPC , the discovery of its participation to the self-renewal of hematopoietic and neural stem cells has cast a new spotlight on its potential role in stem cell biology. However, still little is known on the cellular and molecular mechanisms at play. Here, by combining in vitro and in vivo murine models of PrPC depletion, we establish that PrPC deficiency severely affects the Notch pathway, which plays a major role in neural stem cell maintenance. We document that the absence of PrPC in a neuroepithelial cell line or in primary neurospheres is associated with drastically reduced expression of Notch ligands and receptors, resulting in decreased levels of Notch target genes. Similar alterations of the Notch pathway are recovered in the neuroepithelium of Prnp-/- embryos during a developmental window encompassing neural tube closure. In addition, in line with Notch defects, our data show that the absence of PrPC results in altered expression of Nestin and Olig2 as well as N-cadherin distribution. We further provide evidence that PrPC controls the expression of the epidermal growth factor receptor (EGFR) downstream from Notch. Finally, we unveil a negative feedback action of EGFR on both Notch and PrPC . As a whole, our study delineates a molecular scenario through which PrPC takes part to the self-renewal of neural stem and progenitor cells. Stem Cells 2017;35:754-765.

Regulation of Aquaporin 3 Expression by the AhR Pathway Is Critical to Cell Migration.

The regulation of cell migration is a key factor for the dissemination of metastatic cells during tumor progression. Aquaporins are membrane channels which allow transmembrane fluxes of water and glycerol in cells in a variety of mammalian tissues. Here, we show that AQP3, which has been incriminated in cancer progression, is regulated by the AhR, or dioxin receptor. AhR is a transcription factor which is triggered in response to environmental pollutants and it has been shown to regulate several cellular processes including cell migration and plasticity. In vivo, upon exposure to the aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the expression of AQP3 is increased significantly in several murine tissues including the liver. In vitro, treatment of human HepG2 cells with TCDD also increased the expression of AQP3 mRNA and protein. These effects resulted from the activation of AhR as shown by RNA interference, chromatin immunoprecipitation and the use of several AhR ligands. Immunofluorescence and real-time analysis of cell migration (XCelligence) demonstrated that knockdown of AQP3 mRNA using small interfering RNA impairs the remodeling of cell shape and the triggering of cell migration that is induced by TCDD. Our work reveals, for the first time, a link between exposure to pollutant and the induction of an aquaporin which has been suspected to play a role during metastasis.

Determination of Heavy Metal Concentrations in Normal and Pathological Human Endometrial Biopsies and In Vitro Regulation of Gene Expression by Metals in the Ishikawa and Hec-1b Endometrial Cell Line.

It is well known that several metals, such as lead, mercury, cadmium, and vanadium, can mimic the effects of estrogens (metallo-estrogens). Nevertheless, there are only a few studies that have assessed the effects of toxic metals on the female genital tract and, in particular, endometrial tissue. In this context, we measured the concentrations of several trace elements in human endometrial tissue samples from individuals with hyperplasia or adenocarcinoma and in normal tissues. Hyperplasic endometrial tissue has a 4-fold higher concentration of mercury than normal tissue. Mercury can affect both the AhR and ROS signaling pathways. Thus, we investigated the possible toxic effects of mercury by in vitro studies. We found that mercury increases oxidative stress (increased HO1 and NQO1 mRNA levels) and alters the cytoskeleton in the human endometrial Ishikawa cell line and to a lesser extent, in the "less-differentiated" human endometrial Hec-1b cells. The results might help to explain a potential link between this metal and the occurrence of endometrial hyperplasia.

A generic methodological framework for studying single cell motility in high-throughput time-lapse data.

MOTIVATION: Motility is a fundamental cellular attribute, which plays a major part in processes ranging from embryonic development to metastasis. Traditionally, single cell motility is often studied by live cell imaging. Yet, such studies were so far limited to low throughput. To systematically study cell motility at a large scale, we need robust methods to quantify cell trajectories in live cell imaging data. RESULTS: The primary contribution of this article is to present Motility study Integrated Workflow (MotIW), a generic workflow for the study of single cell motility in high-throughput time-lapse screening data. It is composed of cell tracking, cell trajectory mapping to an original feature space and hit detection according to a new statistical procedure. We show that this workflow is scalable and demonstrates its power by application to simulated data, as well as large-scale live cell imaging data. This application enables the identification of an ontology of cell motility patterns in a fully unsupervised manner. AVAILABILITY AND IMPLEMENTATION: Python code and examples are available online (http://cbio.ensmp.fr/∼aschoenauer/motiw.html)

A head and neck cancer tumor response-specific gene signature for cisplatin, 5-fluorouracil induction chemotherapy fails with added taxanes.

BACKGROUND: It is a major clinical challenge to predict which patients, with advanced stage head and neck squamous cell carcinoma, will not exhibit a reduction in tumor size following induction chemotherapy in order to avoid toxic effects of ineffective chemotherapy and delays for instituting other therapeutic options. Further, it is of interest to know to what extent a gene signature, which identifies patients with tumors that will not respond to a particular induction chemotherapy, is applicable when additional chemotherapeutic agents are added to the regimen. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that predict tumor resistance to induction with cisplatin/5-fluorouracil (PF) or PF and a taxane, we analyzed patient tumor biopsies with whole genome microarrays and quantitative reverse transcriptase-PCR (TLDA) cards. A leave one out cross-validation procedure allowed evaluation of the prediction tool. A ten-gene microarray signature correctly classified 12/13 responders and 7/10 non-responders to PF (92% specificity, 82.6% accuracy). TLDA analysis (using the same classifier) of the patients correctly classified 12/12 responders and 8/10 non-responders (100% specificity, 90.9% accuracy). Further, TLDA analysis correctly predicted the response of 5 new patients and, overall, 12/12 responders and 13/15 non-responders (100% specificity, 92.6% accuracy). The protein products of the genes constituting the signature physically associate with 27 other proteins, involved in regulating gene expression, constituting an interaction network. In contrast, TLDA-based prediction (with the same gene signature) of responses to induction with PF and either of two taxanes was poor (0% specificity, 25% accuracy and 33.3% specificity, 25% accuracy). CONCLUSIONS/SIGNIFICANCE: Successful transfer of the microarray-based gene signature to an independent, PCR-based technology suggests that TLDA-based signatures could be a useful hospital-based technology for determining therapeutic options. Although highly specific for tumor responses to PF induction, the gene signature is unsuccessful when taxanes are added. The results illustrate the subtlety in developing "personalized medicine".

NKT cell-plasmacytoid dendritic cell cooperation via OX40 controls viral infection in a tissue-specific manner.

Invariant natural killer T (iNKT) cells promote immune responses to various pathogens, but exactly how iNKT cells control antiviral responses is unclear. Here, we showed that iNKT cells induced tissue-specific antiviral effects in mice infected by lymphocytic choriomeningitis virus (LCMV). Indeed, iNKT cells inhibited viral replication in the pancreas and liver but not in the spleen. In the pancreas, iNKT cells expressed the OX40 molecule and promoted type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs) through OX40-OX40 ligand interaction. Subsequently, this iNKT cell-pDC cooperation attenuated the antiviral adaptive immune response in the pancreas but not in the spleen. The dampening of pancreatic anti-LCMV CD8(+) T cell response prevented tissue damage in transgenic mice expressing LCMV protein in islet beta cells. Thus, this study identifies pDCs as an essential partner of iNKT cells for mounting an efficient, nondeleterious antiviral response in peripheral tissue.

PPARalpha regulates the hepatotoxic biomarker alanine aminotransferase (ALT1) gene expression in human hepatocytes.

In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) alpha agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPARalpha agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at -574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum.