Caffeine intake exerts dual genome-wide effects on hippocampal metabolism and learning-dependent transcription.

Caffeine is the most widely consumed psychoactive substance in the world. Strikingly, the molecular pathways engaged by its regular consumption remain unclear. We herein addressed the mechanisms associated with habitual (chronic) caffeine consumption in the mouse hippocampus using untargeted orthogonal omics techniques. Our results revealed that chronic caffeine exerts concerted pleiotropic effects in the hippocampus at the epigenomic, proteomic, and metabolomic levels. Caffeine lowered metabolism-related processes (e.g., at the level of metabolomics and gene expression) in bulk tissue, while it induced neuron-specific epigenetic changes at synaptic transmission/plasticity-related genes and increased experience-driven transcriptional activity. Altogether, these findings suggest that regular caffeine intake improves the signal-to-noise ratio during information encoding, in part through fine-tuning of metabolic genes, while boosting the salience of information processing during learning in neuronal circuits.

Mechanisms of innate events during skin reaction following intradermal injection of seasonal influenza vaccine.

The skin plays a crucial role in host defences against microbial attack and the innate cells must provide the immune system with sufficient information to organize these defences. This unique feature makes the skin a promising site for vaccine administration. Although cellular innate immune events during vaccination have been widely studied, initial events remain poorly understood. Our aim is to determine molecular biomarkers of skin innate reaction after intradermal (i.d.) immunization. Using an ex vivo human explant model from healthy donors, we investigated by NanoLC-MS/MS analysis and MALDI-MSI imaging, to detect innate molecular events (lipids, metabolites, proteins) few hours after i.d. administration of seasonal trivalent influenza vaccine (TIV). This multimodel approach allowed to identify early molecules differentially expressed in dermal and epidermal layers at 4 and 18 h after TIV immunization compared with control PBS. In the dermis, the most relevant network of proteins upregulated were related to cell-to-cell signalling and cell trafficking. The molecular signatures detected were associated with chemokines such as CXCL8, a chemoattractant of neutrophils. In the epidermis, the most relevant networks were associated with activation of antigen-presenting cells and related to CXCL10. Our study proposes a novel step-forward approach to identify biomarkers of skin innate reaction. SIGNIFICANCE: To our knowledge, there is no study analyzing innate molecular reaction to vaccines at the site of skin immunization. What is known on skin reaction is based on macroscopic (erythema, redness…), microscopic (epidermal and dermal tissues) and cellular events (inflammatory cell infiltrate). Therefore, we propose a multimodal approach to analyze molecular events at the site of vaccine injection on skin tissue. We identified early molecular networks involved biological functions such cell migration, cell-to-cell interaction and antigen presentation, validated by chemokine expression, in the epidermis and dermis, then could be used as early indicator of success in immunization.

Target exposure and pharmacodynamics study of the indoleamine 2,3-dioxygenase-1 (IDO-1) inhibitor epacadostat in the CT26 mouse tumor model.

Indoleamine-2,3-dioxygénase (IDO1) is an enzyme which converts tryptophan (Trp) into kynurenine (Kyn). Having a critical role in tumor immune escape by decreasing Trp and increasing Kyn levels in the microenvironment, IDO1 was one of the first targets for small molecules drug discovery in the field of immuno-oncology. A potent and selective IDO1 inhibitor such as Epacadostat (EPA) was shown to enhance the antitumor activity by restoring the immune system fitness. As exposure at the site of action and to its specific target are identified as the most important factors for success in drug discovery, the objective of this study was to explore the target exposure and intra-tumor pharmacodynamics effects of EPA drug on the tumor metabolism. To do so, we used both Quantitative Mass Spectrometry Imaging (QMSI) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technologies in order to monitor drug and metabolites distribution and their endogenous quantity in the CT26 mouse tumor model. Target exposure analysis showed that almost 61% of EPA signal (26 µg/g) was concentrated within 38% of the entire tumor surface. Semi quantitative analysis of this region confirmed a positive correlation between IDO1 expression and EPA concentration. In parallel, pharmacodynamics analysis highlighted a response efficacy through Kyn/Trp ratio calculation that was shown decreasing after EPA treatment as noticed in treated CT26 tumors (-82%), plasma (-63%) and blood (-62%) compared to control samples. Finally, 15% and 85% of Kyn signal was found in regions with high and low EPA, respectively. In this study, using QMSI, we went further than only quantifying the metabolites and the drug, by estimating the pharmacological effect efficacy of the drug through a target exposure study handled in different regions of the tumor either expressing IDO1 or Kyn.

Microenvironment Tumor Metabolic Interactions Highlighted by qMSI: Application to the Tryptophan-Kynurenine Pathway in Immuno-Oncology.

Inhibition of NK and effector T-cell functions and activation of regulatory cell populations are the main immunosuppressive effects of indoleamine-2,3-dioxygenase1 (IDO1). By converting tryptophan (Trp) into kynurenine (Kyn), IDO1 is involved in the immune response homeostasis, and its dysregulated expression is described in immune-related pathologies, as tumors that hijack it to evade immune destruction. Thereby, IDO1 inhibitors are being developed to stimulate antitumor immune responses. Existing and standard quantitation methods of IDO1 substrate and metabolite(s) are based on the total level of Trp and its metabolites determined by liquid chromatography tandem mass spectrometry analysis in human plasma, cerebrospinal fluid, and brain. Here, we describe the detection, localization, and absolute quantitation of Trp and Kyn by quantitative mass spectrometry imaging (qMSI) in transfected murine tumor models expressing various levels of IDO1. Myeloid, glycolysis metabolic signatures, and correlation between IDO1 expression and Trp to Kyn conversion are also shown. High-definition IDO1 and GCN2 immunostainings overlaid with Kyn molecular images underline the tumor metabolism and heterogeneity. The development of immunotherapies such as IDO1 inhibitors requires a deep understanding of the immune system, the interplay of cancer cells, and biomarker characterization. Our data underline that qMSI allows the study of the spatial distribution and quantitation of endogenous immune metabolites for biology and pharmacology studies.

QuEChERS sample preparation prior to LC-MS/MS determination of opiates, amphetamines, and cocaine metabolites in whole blood.

Modern LC-MS/MS instruments have sensitivity and scanning velocity high enough to analyze many different compounds in single runs. Consequently, the sample preparation procedure has become the bottleneck for developing efficient, rapid, and cheap multi-compound methods. Here, we examined one-step sample preparation based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) salts to set up and validate a LC-MS/MS method for the simultaneous determination of 35 drugs of abuse and their metabolites in whole blood. Despite large differences in physicochemical properties, this simplified QuEChERS extraction method yielded satisfactory recoveries (until 96%) for the 35 molecules. The amounts of QuEChERS salts had no influence on extraction yield. Chromatographic separation was obtained in less than 6 min. LLOD and LLOQ were 3 and 5 ng/mL, respectively. The procedure was successfully validated and then applied to 253 cases of driving under the influence of drugs (DUID), collected over a 6-month period.