In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; ...however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate 'hepatocyte-like' cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug.
Bile acids (BAs) are recognised as the causative agents of toxicity in drug-induced cholestasis (DIC). Research in isolated mitochondria and HepG2 cells have demonstrated BA-mediated mitochondrial ...dysfunction as a key mechanism of toxicity in DIC. However, HepG2 cells are of limited suitability for DIC studies as they do not express the necessary physiological characteristics. In this study, the mitotoxic potentials of BA mixtures were assessed in isolated mitochondria and a better-suited hepatic model, HepaRG cells. BAs induced structural alterations and a loss of mitochondrial membrane potential (MMP) in isolated mitochondria however, this toxicity did not translate to HepaRG cells. There were no changes in oxygen consumption rate, MMP or ATP levels in glucose and galactose media, indicating that there was no direct mitochondrial toxicity mediated via electron transport chain dysfunction in HepaRG cells. Assessment of key biliary transporters revealed that there was a time-dependent reduction in the expression and activity of multi-drug resistance protein 2 (MRP2), which was consistent with the induction of cytotoxicity in HepaRG cells. Overall, the findings from this study have demonstrated that mitochondrial dysfunction is not a mechanism of BA-induced toxicity in HepaRG cells.
•HepaRG cells are a better suited in vitro model for cholestatic studies than HepG2 cell.•Bile acids cause mitochondrial toxicity in isolated mitochondria but not in HepaRG cells.•Time-dependent alterations in biliary transporters are consistent with the cytotoxicity of bile acid mixtures.•There are important mechanistic differences when bile acids interact at the organelle level versus the whole cell.
Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety ...assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.
Several endothelin receptor antagonists (ERAs) have been developed for the treatment of pulmonary arterial hypertension (PAH). Some of them have been related to clinical cases of hepatocellular ...injury (sitaxentan SIT) and/or cholestasis (bosentan BOS). We aimed to determine if ambrisentan (AMB) and macitentan (MAC), in addition to BOS and SIT, could potentially cause liver damage in man by use of human HepaRG cells. Our results showed that like BOS, MAC-induced cytotoxicity and cholestatic disorders characterized by bile canaliculi dilatation and impairment of myosin light chain kinase signaling. Macitentan also strongly inhibited taurocholic acid and carboxy-2',7'-dichlorofluorescein efflux while it had a much lower inhibitory effect on influx activity compared to BOS and SIT. Moreover, these three drugs caused decreased intracellular accumulation and parallel increased levels of total bile acids (BAs) in serum-free culture media. In addition, all drugs except AMB variably deregulated gene expression of BA transporters. In contrast, SIT was hepatotoxic without causing cholestatic damage, likely via the formation of reactive metabolites and AMB was not hepatotoxic. Together, our results show that some ERAs can be hepatotoxic and that the recently marketed MAC, structurally similar to BOS, can also cause cholestatic alterations in HepaRG cells. The absence of currently known or suspected cases of cholestasis in patients suffering from PAH treated with MAC is rationalized by the lower therapeutic doses and Cmax, and longer receptor residence time compared to BOS.
In this prospective, interventional, international study, we investigate continuous monitoring of hospitalised patients' vital signs using wearable technology as a basis for real-time early warning ...scores (EWS) estimation and vital signs time-series prediction. The collected continuous monitored vital signs are heart rate, blood pressure, respiration rate, and oxygen saturation of a heterogeneous patient population hospitalised in cardiology, postsurgical, and dialysis wards. Two aspects are elaborated in this study. The first is the high-rate (every minute) estimation of the statistical values (e.g., minimum and mean) of the vital signs components of the EWS for one-minute segments in contrast with the conventional routine of 2 to 3 times per day. The second aspect explores the use of a hybrid machine learning algorithm of kNN-LS-SVM for predicting future values of monitored vital signs. It is demonstrated that a real-time implementation of EWS in clinical practice is possible. Furthermore, we showed a promising prediction performance of vital signs compared to the most recent state of the art of a boosted approach of LSTM. The reported mean absolute percentage errors of predicting one-hour averaged heart rate are 4.1, 4.5, and 5% for the upcoming one, two, and three hours respectively for cardiology patients. The obtained results in this study show the potential of using wearable technology to continuously monitor the vital signs of hospitalised patients as the real-time estimation of EWS in addition to a reliable prediction of the future values of these vital signs is presented. Ultimately, both approaches of high-rate EWS computation and vital signs time-series prediction is promising to provide efficient cost-utility, ease of mobility and portability, streaming analytics, and early warning for vital signs deterioration.
Insomnia is a frequent complaint of patients with Parkinson's disease, and it negatively affects quality of life. Drugs that improve both sleep and parkinsonism would be of major benefit to patients ...with Parkinson's disease-related insomnia. We aimed to test the safety and efficacy of subcutaneous night-time only apomorphine infusion in patients with Parkinson's disease and insomnia.
We did a randomised, multicentre, double-blind, placebo-controlled, crossover trial in 11 expert centres in Parkinson's disease and sleep centres in France. Participants aged 35–90 years with fluctuating Parkinson's disease and moderate to severe insomnia (Insomnia Severity Index score ≥15) were randomly assigned to either first receive night-time subcutaneous apomorphine (up to 5 mg/h) or matching placebo. Randomisation was done using a computer-generated plan in blocks of four, stratified by centre. This first intervention was followed by a 14-night washout period, then crossover to the other intervention. The treatment periods consisted of a 10-night titration phase followed by a 7-night fixed-dose phase. The dose was adjusted during the titration phase on the basis of a daily telephone call assessing sleep quality and treatment tolerability. The primary efficacy endpoint was the difference in Parkinson's disease sleep scale (PDSS) scores from the beginning to the end of each treatment period. Analysis was done on an intention-to-treat basis. This trial is registered with ClinicalTrials.gov, NCT02940912.
Between Jan 31, 2017, and Jan 29, 2021, 46 participants were enrolled. 25 (54%) patients were randomly assigned to receive apomorphine first and 21 (46%) patients to receive placebo first. Mean change in PDSS score was significantly greater with night-time apomorphine infusion (15·18 SD 24·34) compared with placebo (5·23 21·52; treatment effect 9·95 95% CI 0·88–19·03; p=0·041). Adverse events were reported in 25 (54%) participants during the apomorphine period and in 17 (37%) participants during the placebo period (p=0·16). Apomorphine was associated with more frequent dizziness than was placebo (seven 15% vs 0; p=0·041).
Subcutaneous night-time only apomorphine infusion improved sleep disturbances according to difference on PDSS score, with an overall safety profile consistent with previous studies in Parkinson's disease. This treatment might be useful to manage sleep disturbances in patients with advanced Parkinson's disease and moderate to severe insomnia.
Orkyn and Aguettant Pharma.
For the French translation of the abstract see Supplementary Materials section.
Prophylaxis with trimethoprim–sulphamethoxazole (TMP-SMZ) is recommended in Toxoplasma-seropositive allogeneic haematopoietic cell transplant (HCT) recipients to prevent reactivation, but it is ...associated with numerous side effects. We report our experience of a pre-emptive approach guided by a polymerase chain reaction (PCR) in patients not receiving prophylaxis.
In this retrospective, single-centre experience, seropositive recipients and seronegative recipients receiving a graft from a seropositive donor were screened by PCR for the presence of Toxoplasma gondii DNA in peripheral blood until at least 6 months after transplantation. Confirmed PCR positivity triggered a pre-emptive anti-Toxoplasma therapy. Cases of Toxoplasma reactivation (using the European Society for Blood and Marrow Transplantation definitions) were compared with four controls (without reactivation), matched in time and recipient serostatus, to identify risk factors for reactivation by multivariate analysis.
From November 2001 to August 2020, 1455 consecutive adult patients (59 cases and 1396 controls) were screened. The overall 1-year cumulative incidence of toxoplasmosis was 4.1% and the 1-year cumulative incidence in the seropositive recipients was 8.8%. Reactivation was associated with second transplant (OR 2.51, 95%CI 1.28–4.94, p 0.011), myeloablative conditioning (OR 2.24, 95%CI 1.17–4.41, p 0.011), total body irradiation (OR 2.29, 95%CI 1.17–4.44, p 0.010), acute graft-versus-host disease (GvHD) (OR 2.27, 95%CI 1.26–4.08, p 0.008) and use of high-dose corticosteroids (OR 2.08, 95%CI 1.14–3.78, p 0.018). In multivariate analysis only acute GvHD remained significant (adjusted OR 2.54, 95%CI 1.16–5.71, p 0.021).
A PCR-based pre-emptive approach might serve as an acceptable alternative for patients unable to start with or to continue TMP-SMZ prophylaxis. Acute GvHD was identified as the single independent predictor for reactivation.
Despite being the gold-standard method for objectively assessing sleep, polysomnography (PSG) faces several limitations as it is expensive, time-consuming, and labor-intensive; requires various ...equipment and technical expertise; and is impractical for long-term or in-home use. Consumer wrist-worn wearables are able to monitor sleep parameters and thus could be used as an alternative for PSG. Consequently, wearables gained immense popularity over the past few years, but their accuracy has been a major concern.
A systematic review of the literature was conducted to appraise the performance of 3 recent-generation wearable devices (Fitbit Charge 4, Garmin Vivosmart 4, and WHOOP) in determining sleep parameters and sleep stages.
Per the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement, a comprehensive search was conducted using the PubMed, Web of Science, Google Scholar, Scopus, and Embase databases. Eligible publications were those that (1) involved the validity of sleep data of any marketed model of the candidate wearables and (2) used PSG or an ambulatory electroencephalogram monitor as a reference sleep monitoring device. Exclusion criteria were as follows: (1) incorporated a sleep diary or survey method as a reference, (2) review paper, (3) children as participants, and (4) duplicate publication of the same data and findings.
The search yielded 504 candidate articles. After eliminating duplicates and applying the eligibility criteria, 8 articles were included. WHOOP showed the least disagreement relative to PSG and Sleep Profiler for total sleep time (-1.4 min), light sleep (-9.6 min), and deep sleep (-9.3 min) but showed the largest disagreement for rapid eye movement (REM) sleep (21.0 min). Fitbit Charge 4 and Garmin Vivosmart 4 both showed moderate accuracy in assessing sleep stages and total sleep time compared to PSG. Fitbit Charge 4 showed the least disagreement for REM sleep (4.0 min) relative to PSG. Additionally, Fitbit Charge 4 showed higher sensitivities to deep sleep (75%) and REM sleep (86.5%) compared to Garmin Vivosmart 4 and WHOOP.
The findings of this systematic literature review indicate that the devices with higher relative agreement and sensitivities to multistate sleep (ie, Fitbit Charge 4 and WHOOP) seem appropriate for deriving suitable estimates of sleep parameters. However, analyses regarding the multistate categorization of sleep indicate that all devices can benefit from further improvement in the assessment of specific sleep stages. Although providers are continuously developing new versions and variants of wearables, the scientific research on these wearables remains considerably limited. This scarcity in literature not only reduces our ability to draw definitive conclusions but also highlights the need for more targeted research in this domain. Additionally, future research endeavors should strive for standardized protocols including larger sample sizes to enhance the comparability and power of the results across studies.
1. Mammalian herbivores can strongly influence nitrogen (N) cycling and herbivore urine could be a central component of the N cycle in grazed ecosystems. Despite its potential role for ecosystem ...productivity and functioning, the fate of N derived from urine has rarely been investigated in grazed ecosystems. 2. This study explored the fate of ¹⁵N-enriched urea in tundra sites that have been either lightly or intensively grazed by reindeer for more than 50 years. We followed the fate of the ¹⁵N applied to the plant canopy, at 2 weeks and 1 year after tracer addition, in the different ecosystem N pools. 3. ¹⁵N-urea was rapidly incorporated in cryptogams and in above-ground parts of vascular plants, while the soil microbial pool and plant roots sequestered only a marginal proportion. Furthermore, the litter layer constituted a large sink for the ¹⁵N-urea, at least in the short term, indicating a high biological activity in the litter in the first phases of organic matter 15 layer and high immobilization decomposition. 4. Mosses and lichens still constituted the largest sink for the ¹⁵N-urea 1 year after tracer addition at both levels of grazing intensity demonstrating their large ability to capture and retain N from urine. Despite large fundamental differences in their traits, deciduous and evergreen shrubs were just as efficient as graminoids in taking up the ¹⁵N-urea. The total recovery of ¹⁵N-urea was lower in the intensively grazed sites, suggesting that reindeer reduce ecosystem N retention. 5. Synthesis. The rapid incorporation of the applied ¹⁵N-urea indicates that arctic plants can take advantage of a pulse of incoming N from urine. In addition, δ¹⁵N values of all taxa in the heavily grazed sites converged towards the δ¹⁵N values for urine, bringing further evidence that urine is an important N source for plants in grazed tundra ecosystems.
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