Impact of interleukin-6 on drug transporters and permeability in the hCMEC/D3 blood-brain barrier model.

The blood-brain barrier (BBB) is a highly selective membrane composed predominantly of brain capillary endothelial cells expressing drug efflux transporters that prevent substrates from accessing the brain. Inflammation is associated with central nervous system diseases and can impair BBB permeability via several mechanisms, including altered transporter and cell junction expression. This can modify the brain's exposure to drugs. However, comprehensive genomic analysis of the impact of interleukin (IL)-6, which plays a key role in the inflammatory response, on the BBB is lacking. In the present study, we analyzed the effects of exposure of hCMEC/D3 cells to 20 ng/mL IL-6 for 72 h. We performed RNA sequencing and ABC transporter efflux assays. Physiologically based pharmacokinetics (PBPK) simulations were conducted to evaluate the potential impact of IL-6 on the digoxin pharmacokinetics profile and brain exposure by decreasing BBB ABCB1 efflux activity. Exposure of hCMEC/D3 cells to IL-6 triggered the deregulation of numerous genes involved in barrier permeability, such as cell junctions, focal adherens complex, and cell adhesion molecules. We observed mild modification of the mRNA expression and efflux activities of ABC transporters. PBPK simulation showed that, if we only consider the impact of IL-6 on ABCB1 transporter, the modification of the digoxin pharmacokinetics profile and brain exposure is slight. IL-6 slightly affected the gene expression levels and activities of ABC transporters on BBB cells, exhibiting a weaker effect than on hepatic cells. However, inflammation may cause other modifications, such as altered BBB permeability, that could modify drug pharmacokinetics.

Impact of Interleukin-6 on Drug-Metabolizing Enzymes and Transporters in Intestinal Cells.

Inflammatory response is characterized by an increase of several cytokines. Some are known to modify drugs pharmacokinetic by reducing the expression levels of drug-metabolizing enzymes (DMEs) and transporters. This impact of inflammatory signaling is well established in hepatic cells, but not in intestinal cells. EpiIntestinal is a 3D human small intestinal tissue model with epithelial polarity, allowing good evaluation of metabolism and drug transport. This study aimed to analyze the effect of IL-6 on this tissue model. RNA sequencing was performed in cells incubated with 5, 10, or 20 ng/mL IL-6 for 8 h to 72 h to study the impact of IL-6 on drug metabolism and pharmacokinetics gene expression. The influence of IL-6 on the activity of cytochromes P450 (CYPs) was studied by measuring metabolite formation of specific substrates with LC-HRMS. Its impact on ATP-binding cassette (ABC) transport was evaluated by measuring intra- and extracellular substrates using spectrofluorometry. Exposure of EpiIntestinal cells to IL-6 resulted in reduction of some CYP mRNAs, such as CYP2C19, CYP2C9, and CYP3A4, by 40% to 50%. Activities of these CYPs were also decreased in EpiIntestinal cells by 20% to > 75%. IL-6 exposure did not modify ABCB1 and ABCCs transporter activities in this model. This study shows that gene expression levels and activities of drug-metabolizing enzymes and ABC transporters may be altered by the pro-inflammatory cytokine IL-6 in intestinal cells. If these results are confirmed in vivo, it may result in pharmacokinetic modifications, such as pre-systemic metabolism, with clinical effects, and require dosage adaptation.

Structure-activity relationship study: Mechanism of cyto-genotoxicity of Nitropyrazole-derived high energy density materials family.

Stringent toxicological tests have to be performed prior to the industrial development of alternative chemicals particularly high energy dense materials (HEDMs) such as explosives. The properties (e.g., power, stability) of these compounds are constantly being improved, the current axis of research being the nitration of nitrogen heterocycles leading to HEDMs such as nitropyrazole-derived molecules. However, except for 3,4,5-trinitropyrazole (3,4,5-TNP), which was shown to be highly toxic in mice, the toxicological impact of these HEDMs has so far not been investigated. Furthermore, as industrials are strongly advised to develop alternative safety testing assays to in vivo experiments, we herein focused on determining the cytotoxic and genotoxic effects of seven Nitropyrazole-derived HEDMs on three rodent cell lines (mouse embryonic BALB/3T3 clone A31 cells, Chinese hamster ovary cells CHO-K1 and mouse lymphoma L5178Y TK +/- clone (3.7.2C) cells), two human fibroblast lines (CRC05, PFS04062) and on the human hepatic HepaRG model (both in proliferative and differentiated cells). A stronger cytotoxic effect was observed for 1,3-dinitropyrazole (1, 3-DNP) and 3,4,5-TNP in all cell lines, though differentiated HepaRG cells clearly displayed fewer likely due to the metabolism and elimination of these molecules by their functional biotransformation pathways. At the mechanistic level, the sub-chronic cytotoxic and genotoxic effects were linked to ROS/RNS production (experimental assays), HA2.X and to transcriptomic data highlighting the increase in DNA repair mechanisms.

Severe hemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing.

Essentials No F8 genetic abnormality is detected in about 2% of severe hemophilia A patients. Detection of F8 structural variants remains a challenge. We identified a new F8 rearrangement in a severe hemophilia A patient using nanopore sequencing. We highlight the value of single-molecule long-read sequencing technologies in a genomics laboratory. BACKGROUND: No F8 genetic abnormality is detected in about 2% of severe hemophilia A patients using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. OBJECTIVE: To characterize, in a genetically unresolved severe hemophilia A patient, a new Xq28 rearrangement disrupting F8 using comprehensive molecular techniques including nanopore sequencing. RESULTS: Long-range polymerase chain reaction (PCR) performed throughout F8 identified a nonamplifiable region in intron 25 indicating the presence of a genomic rearrangement. F8 messanger ribonucleic acid (mRNA) analysis including 3'rapid amplification of complementary deoxyribonucleic acid (cDNA) ends and nanopore sequencing found the presence of a F8 fusion transcript in which F8 exon 26 was replaced by a 742-bp pseudoexon corresponding to a noncoding region located at the beginning of the long arm of chromosome X (Xq12; chrX: 66 310 352-66 311 093, GRCh37/hg19). Cytogenetic microarray analysis found the presence of a Xq11.1q12 gain of 3.8 Mb. The PCR amplification of junction fragments and fluorescent in situ hybridization (FISH) analysis found that the Xq11q12 duplicated region was inserted in the F8 intron 25 genomic region. CONCLUSION: We characterized a novel genomic rearrangement in which a 3.8-Mb Xq11.1q12 gain inserted in the F8 intron 25 led to an aberrant fusion transcript in a patient with severe hemophilia A (HA), using comprehensive molecular techniques. This study highlights the value of single-molecule long-read sequencing technologies for molecular diagnosis of HA especially when conventional genetic approaches have failed.

Metagenomic Next-Generation Sequencing Reveals Individual Composition and Dynamics of Anelloviruses during Autologous Stem Cell Transplant Recipient Management.

Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence.

Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy).

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5-1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%. Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results.

The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline.

BACKGROUND: Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples. RESULTS: We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart). CONCLUSION: The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.