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IMPORTANCE:. Mechanical power (MP) could serve as a valuable parameter in clinical practice to estimate the likelihood of adverse outcomes. However, the safety thresholds for MP in mechanical ventilation remain underexplored and contentious. OBJECTIVES:. This study aims to investigate the association between MP and hospital mortality across varying degrees of lung disease severity, classified by Pao2/Fio2 ratios. DESIGN, SETTING, AND PARTICIPANTS:. This is a retrospective cohort study using automatically extracted data. Patients admitted to the ICU of a tertiary referral hospital in The Netherlands between 2018 and 2024 and ventilated in pressure-controlled mode were included. MAIN OUTCOMES AND MEASURES:. Logistic regression, adjusted for age, sex, Acute Physiology and Chronic Health Evaluation-IV score, and Pao2/Fio2 ratio, was used to calculate the odds ratio (OR) for all-cause in-hospital mortality. RESULTS:. A total of 2184 patients were analyzed, with a mean age of 62.5 ± 13.8 years, of whom 1508 (70.2%) were male. The mean MP was highest in patients with the lowest Pao2/Fio2 ratios (21.5 ± 6.5 J/min) compared with those with the highest ratios (12.0 ± 3.8 J/min; p < 0.001). Adjusted analyses revealed that increased MP was associated with higher mortality (OR, 1.06; 95% CI, 1.03–1.09 per J/min increase). Similarly, MP normalized for body weight showed a stronger association with mortality (OR, 1.004; 95% CI, 1.002–1.006 per J/min/kg increase). An increase in mortality was seen when MP exceeded 16–18 J/min. CONCLUSIONS AND RELEVANCE:. Our findings demonstrate a significant association between MP and hospital mortality, even after adjusting for key confounders. Mortality increases notably when MP exceeds 16–18 J/min. Normalized MP presents an even stronger association with mortality. These results underscore the need for further research into ventilation strategies that consider MP adjustments.

Background/Objectives: The pulmonary artery pulsatility index (PAPi, calculated as (SPAP − DPAP)/mRAP) has been suggested as a measure of right ventricular–vascular coupling (RVVC) and as a prognostic parameter in cardiovascular conditions, particularly right ventricular failure. This retrospective study investigated the relationship between the PAPi and its components with disease severity parameters, the RVVC, and clinical outcomes in children with pulmonary arterial hypertension (PAH). Methods: We analyzed data from 111 children from the Dutch National Registry with PAH. The PAPi (median 6.0 [3.9–8.3]) was calculated from heart catheterization data and the RVVC was determined as the TAPSE/sPAP ratio via echocardiography (0.25 ± 0.12 mm/mmHg). Disease severity was characterized by clinical, hemodynamic, and laboratory data. Cox proportional hazard modeling assessed the PAPi’s predictive value for transplant-free survival. Results: There was no correlation between the RVVC and PAPi (R = −0.208, p = 0.111, n = 60). The PAPi correlated negatively with uric acid (R = −0.387, p < 0.001) but not with other disease severity parameters. Mean right atrial pressure correlated with multiple disease severity indicators. Transplant-free survival rates at 1, 3, and 5 years were 87%, 79%, and 73%, respectively. Neither the PAPi nor its components correlated with transplant-free survival. Conclusions: In conclusion, the PAPi does not correlate with the RVVC and this study could not demonstrate any prognostic value of the PAPi regarding disease severity or outcomes in children with PAH, challenging its utility in this population.

Processed milk and milk products produced from bovine milk, commonly contain β-casein A1 (βCA1) and β-casein A2 (βCA2). Since the presence of βCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains βCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between βCA2 and βCA1, was developed and real-life applicability was shown on raw milk samples from genotyped A1A1, A1A2 and A2A2 cows. Because of its ability to discriminate between βCA2 and βCA1, this newly developed method was able to detect the addition of common bovine A1A2 milk to A2A2 milk, as low as 1%. Besides the detection of A2A2 milk purity, the developed assay can also be implemented as a rapid phenotyping method at dairy farms to replace the more invasive DNA-based screening. Additionally, the developed method was capable of detecting the addition of common bovine milk up to 1% in sheep, goat, buffalo, horse and donkey milk, which conforms to EU recommendations. In conclusion, a newly developed multiplex method capable of reliably detecting the dilution of A2A2 milk of multiple species, with common bovine milk up to 1%, is presented.

Coeliac disease (CeD) is an immune-mediated disorder characterised by gluten-triggered inflammation and damage in the small intestine, with lifelong gluten-free diet (GFD) as the only treatment. It is a multifactorial disease, involving genetic and environmental susceptibility factors, and its complexity and lack of comprehensive human model systems have hindered understanding of its pathogenesis and development of new treatments. Therefore, it is crucial to establish systems that recapitulate patient genetic background and the interactions between the small intestinal epithelial barrier, immune cells, and environment that contribute to CeD. In this review, we discuss disease complexity, recent advances in stem cell biology, organoids, tissue co-cultures, and organ-on-chip (OoC) systems that facilitate the development of comprehensive human model systems, and model applications in preclinical studies of potential treatments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The Wilms' tumor protein 1 (WT1) is a well-known and prioritized tumor-associated antigen expressed in numerous solid and blood tumors. Its abundance and immunogenicity have led to the development of different WT1-specific immune therapies. The driving player in these therapies, the WT1-specific T-cell receptor (TCR) repertoire, has received much less attention. Importantly, T cells with high affinity against the WT1 self-antigen are normally eliminated after negative selection in the thymus and are thus rare in peripheral blood. Here, we developed computational models for the robust and fast identification of WT1-specific TCRs from TCR repertoire data. To this end, WT1 37-45 (WT1-37) and WT1 126-134 (WT1-126)-specific T cells were isolated from WT1 peptide-stimulated blood of healthy individuals. The TCR repertoire from these WT1-specific T cells was sequenced and used to train a pattern recognition model for the identification of WT1-specific TCR patterns for the WT1-37 or WT1-126 epitopes. The resulting computational models were applied on an independent published dataset from acute myeloid leukemia (AML) patients, treated with hematopoietic stem cell transplantation, to track WT1-specific TCRs in silico. Several WT1-specific TCRs were found in AML patients. Subsequent clustering analysis of all repertoires indicated the presence of more diverse TCR patterns within the WT1-specific TCR repertoires of AML patients in complete remission in contrast to relapsing patients. We demonstrate the possibility of tracking WT1-37 and WT1-126-specific TCRs directly from TCR repertoire data using computational methods, eliminating the need for additional blood samples and experiments for the two studied WT1 epitopes., Competing Interests: Declarations. Ethics approval and consent to participate: The use of human samples in this study was approved by the Ethics Committee of the Antwerp University Hospital and the University of Antwerp under reference number 15/19/210. Consent for publication: Not applicable. Competing interests: K.L., P.M. and B.O. hold shares in ImmuneWatch BV, an immunoinformatics company., (© 2024. The Author(s).)

Background: Data quality is fundamental to maintaining the trust and reliability of health data for both primary and secondary purposes. However, before the secondary use of health data, it is essential to assess the quality at the source and to develop systematic methods for the assessment of important data quality dimensions., Objective: This case study aims to offer a dual aim-to assess the data quality of height and weight measurements across 7 Belgian hospitals, focusing on the dimensions of completeness and consistency, and to outline the obstacles these hospitals face in sharing and improving data quality standards., Methods: Focusing on data quality dimensions completeness and consistency, this study examined height and weight data collected from 2021 to 2022 within 3 distinct departments-surgical, geriatrics, and pediatrics-in each of the 7 hospitals., Results: Variability was observed in the completeness scores for height across hospitals and departments, especially within surgical and geriatric wards. In contrast, weight data uniformly achieved high completeness scores. Notably, the consistency of height and weight data recording was uniformly high across all departments., Conclusions: A collective collaboration among Belgian hospitals, transcending network affiliations, was formed to conduct this data quality assessment. This study demonstrates the potential for improving data quality across health care organizations by sharing knowledge and good practices, establishing a foundation for future, similar research., (© Jens Declerck, Bert Vandenberk, Mieke Deschepper, Kirsten Colpaert, Lieselot Cool, Jens Goemaere, Mona Bové, Frank Staelens, Koen De Meester, Eva Verbeke, Elke Smits, Cami De Decker, Nicky Van Der Vekens, Elin Pauwels, Robert Vander Stichele, Dipak Kalra, Pascal Coorevits. Originally published in JMIR Medical Informatics (https://medinform.jmir.org).)

This study focuses on refining growth-rate-based drug response metrics for patient-derived tumor organoid screening using brightfield live-cell imaging. Traditional metrics like Normalized Growth Rate Inhibition (GR) and Normalized Drug Response (NDR) have been used to assess organoid responses to anticancer treatments but face limitations in accurately quantifying cytostatic and cytotoxic effects across varying growth rates. Here, we introduce the Normalized Organoid Growth Rate (NOGR) metric, specifically developed for brightfield imaging-based assays. A label-free image analysis model was applied to segment organoids precisely, track their growth rates over time, and classify viable and dead organoids. Testing eleven phenotypically distinct pancreatic cancer organoid models with five chemotherapeutics demonstrates that the NOGR metric more effectively captures cytostatic and cytotoxic drug effects compared to existing methods. This approach enhances the biological relevance of drug sensitivity assessments on organoids and offers a valuable tool for advancing personalized cancer treatment strategies., Competing Interests: Competing interests: The authors declare the following competing interests: C.D., M.L.C., E.C.D.L.H., and A.L. are co-founders of Orbits® Oncology, an image and data analysis platform that was used in this study. Ethical approval: Pancreatic ductal adenocarcinoma (PDAC) samples were obtained from three expert clinical centers under the PaCaOmics clinical trial (number 2011-A01439-32) after receiving ethics review board approval. Consent forms of informed patients were collected and registered in a central database., (© 2024. The Author(s).)

Cold atmospheric plasma (CAP) is a tool with the ability to generate reactive oxygen and nitrogen species (RONS), which can induce therapeutic effects like disinfection, wound healing, and cancer treatment. In the plasma oncology field, CAP-treated hydrogels (PTHs) are being explored for the local administration of CAP-derived RONS as a novel anticancer approach. PTHs have shown anticancer effects in vitro, however, they have not yet been studied in more relevant cancer models. In this context, the present study explores for the first time the therapeutic potential of PTHs using an advanced in ovo cancer model. PTHs composed of alginate (Alg), gelatin (Gel), Alg/Gel combination, or Alg/hyaluronic acid (HA) combination are investigated. All embryos survived the PTHs treatment, suggesting that the in ovo model could become a time- and cost-effective tool for developing hydrogel-based anticancer approaches. Results revealed a notable reduction in CD44+ cell population and their proliferative state for the CAP-treated Alg-HA condition. Moreover, the CAP-treated Alg-HA formulation alters the extracellular matrix composition, which may help combat drug-resistance. In conclusion, the present study validates the utility of in ovo cancer model for PTHs exploration and highlights the promising potential of Alg-based PTHs containing HA and CAP-derived RONS for cancer treatment., (© 2024 The Author(s). Macromolecular Bioscience published by Wiley‐VCH GmbH.)

Non-thermal plasma (NTP) shows promise as a potent anti-cancer therapy with both cytotoxic and immunomodulatory effects. In this study, we investigate the chemical and biological effects of NTP-induced oxidation on several key, determinant immune checkpoints of natural killer (NK) cell function. We used molecular dynamics (MD) and umbrella sampling simulations to investigate the effect of NTP-induced oxidative changes on the MHC-I complexes HLA-Cw4 and HLA-E. Our simulations indicate that these chemical alterations do not significantly affect the binding affinity of these markers to their corresponding NK cell receptor, which is supported with experimental read-outs of ligand expression on human head and neck squamous cell carcinoma cells after NTP application. Broadening our scope to other key ligands for NK cell reactivity, we demonstrate rapid reduction in CD155 and CD112, target ligands of the inhibitory TIGIT axis, and in immune checkpoint CD73 immediately after treatment. Besides these transient chemical alterations, the reactive species in NTP cause a cascade of downstream cellular reactions. This is underlined by the upregulation of the stress proteins MICA/B, potent ligands for NK cell activation, 24 h post treatment. Taken together, this work corroborates the immunomodulatory potential of NTP, and sheds light on the interaction mechanisms between NTP and cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The planned missions to the Moon and Mars will present more significant health challenges to astronauts compared to low earth orbit missions. During deep space missions, astronauts will be constantly exposed to Space radiation (SR). Multiple rodent studies suggest that < 25 cGy of SR impairs performance in executive functions, which play a key role in advanced cognitive processes, but also regulate response inhibition and impulse control. There is the possibility that SR exposure may exacerbate aberrant behaviors evoked by psychological stress related to exposure to isolated and confined (ICE) hostile environment or independently induce additional aberrant behaviors. This study has determined that female Wistar rats exposed to 10 cGy of 250 MeV/n He had an increased risk taking propensity (RTP)\compared to shams. The increased RTP of the He-exposed rats was associated with significantly increased reaction times during the trials, suggesting a SR-induced loss of processing speed. The response times of the He-exposed rats were even further reduced in trials that immediately followed a loss, raising the possibility that conflict and interference avoidance may be impaired after SR exposure. Whether these findings occur following other types of SR exposure, and/or in male rats remains to be determined., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare in relation to the studies reported in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Non-thermal plasma (NTP) has shown promising anti-cancer effects, but there is still limited knowledge about the underlying cell death mechanisms induced by NTP and inherent differences between NTP treatment modalities. This study aimed to investigate four major regulated cell death (RCD) pathways, namely apoptosis, pyroptosis, necroptosis, and ferroptosis, in melanoma cancer cells following NTP treatment, and to provide an overview of molecular mechanistic differences between direct and indirect NTP treatment modalities. To discriminate which cell death pathways were triggered after treatment, specific inhibitors of apoptosis, pyroptosis, necroptosis, and ferroptosis were evaluated. RCD-specific molecular pathways were further investigated to validate the findings with inhibitors. Both direct and indirect NTP treatment increased caspase 3/7 and annexin V expression, indicative of apoptosis, as well as lipid peroxidation, characteristic of ferroptosis. Pyroptosis, on the other hand, was only induced by direct NTP treatment, evidenced by increased caspase 1 activity, whereas necroptosis was stimulated in a cell line-dependent manner. These findings highlight the molecular differences and implications of direct and indirect NTP treatment for cancer therapy. Altogether, activation of multiple cell death pathways offers advantages in minimizing treatment resistance and enhancing therapeutic efficacy, particularly in a combination setting. Understanding the mechanisms underlying NTP-induced RCD will enable the development of strategic combination therapies targeting multiple pathways to achieve cancer lethality., (© 2024. The Author(s).)

Here, we present a protocol to perform CRISPR-Cas9 genome editing in human resting primary natural killer (NK) and NK-92 cells. We describe steps for guide RNA selection, ribonucleoprotein (RNP) complex formation, delivery via Nucleofection, and analysis of CRISPR edits to assess editing efficiencies. This protocol offers a tool for functional studies in NK cells, paving the way for potential applications in immunotherapy and beyond. We also discuss limitations such as off-target effects and cell-type-specific considerations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To evaluate the effect of combining pain education and virtual reality (VR) exposure therapy using a cognitive-behavioral therapy-informed approach (virtual reality-based cognitive behavioral therapy [VR-CBT]) on pain intensity, fear of movement, and trunk movement in individuals with persistent low back pain., Materials and Methods: Thirty-seven participants were recruited in a single cohort repeated measures study, attending 3 sessions 1 week apart. The VR-CBT intervention included standardized pain education (session 1) and virtual reality-based exposure therapy (VRET; session 2) incorporating gameplay with mixed reality video capture and reflective feedback of performance. Outcome measures (pain intensity, pain-related fear of movement (Tampa Scale of Kinesiophobia), and trunk kinematics during functional movements (maximum amplitude and peak velocity) were collected at baseline (session 1) and 1 week after education (session 2) and VRET (session 3). One-way repeated measures analysis of variances evaluated change in outcomes from baseline to completion. Post hoc contrasts evaluated effect sizes for the education and VR components of VR-CBT., Results: Thirty-four participants completed all sessions. Significant ( P < 0.001) reductions were observed in mean (SD) pain (baseline 5.9 [1.5]; completion 4.3 [2.1]) and fear of movement (baseline 42.6 [6.4]; completion 34.3 [7.4]). Large effect sizes (Cohen d ) were observed for education (pain intensity: 0.85; fear of movement: 1.28), whereas the addition of VRET demonstrated very small insignificant effect sizes (pain intensity: 0.10; fear of movement: 0.18). Peak trunk velocity, but not amplitude, increased significantly ( P < 0.05) across trunk movement tasks., Conclusion: A VR-CBT intervention improved pain, pain-related fear of movement, and trunk kinematics. Further research should explore increased VR-CBT dosage and mechanisms underlying improvement., Competing Interests: This project was completed with funding support through the Royal Brisbane and Women’s Hospital (RBWH) Foundation and the Physiotherapy Department at RBWH, Brisbane, Australia. The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Current guidelines for the prevention and management of cardiovascular diseases (CVD) provide similar recommendations for the use of statins in both women and men. In this study, we assessed sex differences in the intensity of statin prescriptions at initiation and in the achievement of treatment targets, among individuals without and with CVD, in a primary care setting., Methods: Electronic health record data from statin users were extracted from the PHARMO Data Network. Poisson regressions were used to investigate sex differences in statin intensity and in the achievement of treatment targets. Analyses were stratified by age group, disease status and/or CVD risk category., Results: We included 82 714 individuals (46% women) aged 40-99 years old. In both sexes, the proportion of individuals with a dispensed prescription for high-intensity statin at initiation increased between 2011 and 2020. Women were less likely to be prescribed high-intensity statins as compared with men, both in the subgroups without a history of CVD (risk ratio (RR) 0.69 (95% CI: 0.63 to 0.75)) and with CVD (RR 0.77 (95% CI: 0.74 to 0.81)). Women were less likely than men to achieve target levels of low-density lipoprotein cholesterol following statin initiation in the subgroup without CVD (RR 0.98 (95% CI: 0.97 to 1.00)) and with a history of CVD (RR 0.94 (95% CI: 0.89 to 0.98))., Conclusion: Compared with men, women were less likely to be prescribed high-intensity statins at initiation and to achieve treatment targets, both in people without and with a history of CVD, and independent of differences in other individual and clinical characteristics., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background Physical activity has health benefits for adults with acquired brain injury, but it is a challenge to increase physical activity during inpatient rehabilitation. The objectives of this pilot study were to determine whether a physiotherapy-supervised inpatient walking program was feasible and able to improve physical activity and sedentary behaviour in the short and medium term. Methods Adults with acquired brain injury receiving inpatient rehabilitation undertook twice-weekly supervised walks plus behavioural therapy for 4 weeks. Feasibility was measured via recruitment, participation and drop out rates, adverse events and intervention delivery costs. Physical activity and sedentary behaviour were measured with an activPAL. Assessments were conducted at baseline, post-intervention and 3-6 months post-intervention. Results The program was safe to deliver (no adverse events), recruitment rate was 55% (16/29) and the participation rate for eligible individuals was high (14/19, 74%). However, the program had a high drop out rate (7/16, 44%) and physical activity and sedentary behaviour did not significantly change during the 4-week intervention. Costs were AU$427.71/participant. Physical activity and sedentary behaviour did improve 3-6 months after the intervention (vs baseline, on average: +3913 steps per day, 95% CI: 671, 7156). Conclusion This pilot study demonstrated a supervised physiotherapy walking program is safe and feasible to recruit in an inpatient setting. However, drop out during the study was high and behaviour change did not occur. More work is required to boost physical activity during sub-acute rehabilitation for acquired brain injury.

The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4 + T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation., Competing Interests: Declaration of interests K.L., P.M., and B.O. are co-founders, board directors, and shareholders of ImmuneWatch. None of the work presented here was influenced in any way by this. ImmuneWatch had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To describe opportunities and challenges experienced from the four pharmacoepidemiological database studies included in the rivaroxaban post authorisation safety study (PASS) programme and propose ways to maximise the value of population-based observational research when addressing regulatory requirements., Design: PASS programme of rivaroxaban carried out as part of the regulatory postapproval commitment to the European Medicines Agency., Setting: Clinical practice in Germany, the Netherlands, Sweden and the UK (electronic health records)-undertaken by pharmacoepidemiology research teams using country-specific databases with different coding structures., Participants: 355 152 patients prescribed rivaroxaban and 338 199 patients prescribed vitamin K antagonists., Results: Two major challenges that were encountered throughout the lengthy PASS programme were related to: (1) finalising country-tailored study designs before the extent of rivaroxaban uptake was known, and (2) new research questions that arose during the programme (eg, those relating to an evolving prescribing landscape)., Recommendations: We advocate the following strategies to help address these major challenges (should they arise in any future PASS): conducting studies based on a common data model that enable the same analytical tools to be applied when using different databases; maintaining early, clear, continuous communication with the regulator (including discussing the potential benefit of studying drug use as a precursor to planning a safety study); consideration of adaptive designs whenever uncertainty exists and following an initial period of data collection; and setting milestones for the review of study objectives., Competing Interests: Competing interests: GB was an employee of Bayer AB, Sweden, at the time the PASS programme was conducted and has received consultancy fees from Bayer AG, Germany, thereafter as an external consultant. YB, CT, KS-W, MS-G, PV, TD, GN and PA are current employees of Bayer AG, Germany. MH was an employee of Bayer AG, Germany, at the time the PASS programme was conducted. AR and LAGR work for Spanish Centre for Pharmacoepidemiological Research, Spain, which received research funding for the study carried out within the rivaroxaban PASS programme. TS is an employee of Leibniz Institute for Prevention Research and Epidemiology, Germany, which received research funding for the study carried out within the rivaroxaban PASS programme. AV was an employee at BIPS at the time when the study was conducted. RH, ES and KMAS work for PHARMO Institute for Drug Outcomes Research, Netherlands, which received research funding for the study carried out within the rivaroxaban PASS programme. LF works for Friberg Research AB, Sweden, which received research funding for the study carried out within the rivaroxaban PASS programme., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: This study explores the repurposing of Auranofin (AF), an anti-rheumatic drug, for treating non-small cell lung cancer (NSCLC) adenocarcinoma and pancreatic ductal adenocarcinoma (PDAC). Drug repurposing in oncology offers a cost-effective and time-efficient approach to developing new cancer therapies. Our research focuses on evaluating AF's selective cytotoxicity against cancer cells, identifying RNAseq-based biomarkers to predict AF response, and finding the most effective co-therapeutic agents for combination with AF., Methods: Our investigation employed a comprehensive drug screening of AF in combination with eleven anticancer agents in cancerous PDAC and NSCLC patient-derived organoids (n = 7), and non-cancerous pulmonary organoids (n = 2). Additionally, we conducted RNA sequencing to identify potential biomarkers for AF sensitivity and experimented with various drug combinations to optimize AF's therapeutic efficacy., Results: The results revealed that AF demonstrates a preferential cytotoxic effect on NSCLC and PDAC cancer cells at clinically relevant concentrations below 1 µM, sparing normal epithelial cells. We identified Carbonic Anhydrase 12 (CA12) as a significant RNAseq-based biomarker, closely associated with the NF-κB survival signaling pathway, which is crucial in cancer cell response to oxidative stress. Our findings suggest that cancer cells with low CA12 expression are more susceptible to AF treatment. Furthermore, the combination of AF with the AKT inhibitor MK2206 was found to be particularly effective, exhibiting potent and selective cytotoxic synergy, especially in tumor organoid models classified as intermediate responders to AF, without adverse effects on healthy organoids., Conclusion: Our research offers valuable insights into the use of AF for treating NSCLC and PDAC. It highlights AF's cancer cell selectivity, establishes CA12 as a predictive biomarker for AF sensitivity, and underscores the enhanced efficacy of AF when combined with MK2206 and other therapeutics. These findings pave the way for further exploration of AF in cancer treatment, particularly in identifying patient populations most likely to benefit from its use and in optimizing combination therapies for improved patient outcomes., (© 2024. The Author(s).)

Background: The European rivaroxaban post-authorization safety study evaluated bleeding risk among patients initiated on rivaroxaban or vitamin K antagonists for the treatment and secondary prevention of venous thromboembolism in routine clinical practice., Methods: Cohorts were created using electronic healthcare databases from the UK, the Netherlands, Germany and Sweden. Patients with a first prescription of rivaroxaban or vitamin K antagonist during the period from December 2011 (in the UK, January 2012) to December 2017 (in Germany, December 2016) for venous thromboembolism indication, with no record of atrial fibrillation or recent cancer history, were observed until the occurrence of each safety outcome (hospitalization for intracranial, gastrointestinal, urogenital or other bleeding), death or study end (December 2018; in Germany, December 2017). Crude incidence rates of each outcome per 100 person-years were computed., Results: Overall, 44 737 rivaroxaban and 45 842 vitamin K antagonist patients were enrolled, mean age, 59.9-63.8 years. Incidence rates were similar between rivaroxaban and vitamin K antagonist users with some exceptions, including higher incidence rates for gastrointestinal bleeding in rivaroxaban users than in vitamin K antagonist users. Among rivaroxaban users, mortality and bleeding risk generally increased with age, renal impairment and diabetes., Conclusions: This study provides further data from routine clinical practice that broadly support safety profile of rivaroxaban for VTE indication and complement findings from previous randomized clinical trials., Competing Interests: Competing interests Ana Ruigómez and Luis Alberto García Rodríguez work for CEIFE, which has received research grants from Bayer AG; Tania Schink works and Annemarie Voss worked for the Leibniz Institute for Prevention Research and Epidemiology – BIPS, an independent, non-profit research institute, which occasionally conducts post-authorization safety studies (PASS) requested by health authorities, financed by the pharmaceutical industry and performed in line with the ENCePP Code of Conduct; Ron M. C. Herings, Elisabeth Smits and Karin Swart-Polinder are employees of the PHARMO Institute for Drug Outcomes Research, an independent research institute which performs financially supported studies for government and related healthcare authorities and several pharmaceutical companies; Yanina Balabanova and Kiliana Suzart-Woischnik are employees of Bayer AG; and Gunnar Brobert was an employee of Bayer AB at the time of the studies., (Copyright: © 2024 Ruigómez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plasmodium vivax malaria is increasingly recognized as a major global health problem and the socio-economic impact of P.vivax-induced burden is huge. Vaccine development against P. vivax malaria has been hampered by the lack of an in vitro culture system and poor access to P. vivax sporozoites. The recent generation of Plasmodium falciparum parasites that express a functional P. vivax AMA1 molecule has provided a platform for in vitro evaluation of PvAMA1 as a potential blood stage vaccine. Three so-called PvAMA1 Diversity Covering (DiCo) proteins were designed to assess their potential to induce a functional and broad humoral immune response to the polymorphic PvAMA1 molecule. Rabbits were immunized with the mixture of three, Pichia-produced, PvAMA1 DiCo proteins, as well as with 2 naturally occurring PvAMA1 alleles. For these three groups, the experimental adjuvant raffinose fatty acid sulfate ester (RFASE) was used, while in a fourth group the purified main mono-esterified constituent (RSL10) of this adjuvant was used. Animals immunized with the mixture of the three PvAMA1 DiCo proteins in RFASE showed high anti-PvAMA1 antibody titers against three naturally occurring PvAMA1variants while also high growth-inhibitory capacity was observed against P. falciparum parasites expressing PvAMA1. This supports further clinical development of the PvAMA1 DiCo mixture as a potential malaria vaccine. However, as the single allele PvAMA1 SalI-group showed similar characteristics in antibody titer and inhibition levels as the PvAMA1 DiCo mixture-group, this raises the question whether a mixture is really necessary to overcome the polymorphism in the vaccine candidate. RFASE induced strong humoral responses, as did the animals immunized with the purified component, RSL10. This suggests that RSL10 is the active ingredient. However, one of the RSL10-immunized animal showed a delayed response, necessitating further research into the clinical development of RSL10., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: It remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma., Methods: RNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70 + tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs., Results: In this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70 + tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70 + tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner., Conclusions: We disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70 + cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors., (© 2024. The Author(s).)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Objectives: To review cut-points calibrated and independently validated from wrist-worn ActiGraph accelerometers to measure moderate to vigorous physical activity (MVPA) and time spent sedentary (SED) in children and adolescents., Design: Systematic literature review., Methods: Five databases were searched for relevant cut-point calibration and independent validation studies relating to wrist worn ActiGraphs in children and adolescents from inception through 30 April 2022. Extracted data included: country of publication; study name; population; device model; wear location; sampling frequency; epoch length; activity protocol; criterion method and definitions used to classify PA intensity; statistical methods for calibration; statistical methods for validation/cross-validation; and MVPA and SED outcome., Results: Fourteen calibration studies and seven independent validation studies were identified. Calibrated cut-points for MVPA vector magnitude counts ranged from 7065 to 9204 counts per minute (cpm) and 63.5 to 201 milli-gravitational units (mg). For SED, calibrated cut-points ranged from <2556 cpm to 4350 cpm and 30.8 to 48.1 mg. Classification accuracy values determined by independent validation studies varied, with kappa values ranging from 0.31 to 0.60 and area under the curve statistics ranging from 0.51 to 0.84 for MVPA and kappa values ranging from 0.31 to 0.44 and area under the curve statistics ranging from 0.70 to 0.85 for SED., Conclusions: The results of this systematic literature review support the use of the Crouter and colleagues cut-points for the measurement of MVPA and SED for children and adolescents aged 6-12 years. Further work is required to independently validate cut-points developed in younger children and older adolescents., Competing Interests: Declaration of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Herein we report a new library of 2,3-pyrrolidinedione analogues that expands on our previous report on the antimicrobial studies of this heterocyclic scaffold. The novel 2,3-pyrrolidinediones reported herein have been evaluated against S. aureus and methicillin-resistant S. aureus (MRSA) biofilms, and this work constitutes our first report on the antibiofilm properties of this class of compounds. The antibiofilm activity of these 2,3-pyrrolidinediones has been assessed through minimum biofilm eradication concentration (MBEC) and minimum biofilm inhibition concentration (MBIC) assays. The compounds displayed antibiofilm properties and represent intriguing scaffolds for further optimization and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Discovery of epitope-specific T-cell receptors (TCRs) for cancer therapies is a time consuming and expensive procedure that usually requires a large amount of patient cells. To maximize information from and minimize the need of precious samples in cancer research, prediction models have been developed to identify in silico epitope-specific TCRs. In this chapter, we provide a step-by-step protocol to train a prediction model using the user-friendly TCRex webtool for the nearly universal tumor-associated antigen Wilms' tumor 1 (WT1)-specific TCR repertoire. WT1 is a self-antigen overexpressed in numerous solid and hematological malignancies with a high clinical relevance. Training of computational models starts from a list of known epitope-specific TCRs which is often not available for new cancer epitopes. Therefore, we describe a workflow to assemble a training data set consisting of TCR sequences obtained from WT1 37-45 -reactive CD8 T cell clones expanded and sorted from healthy donor peripheral blood mononuclear cells., Competing Interests: Conflicts of interest K.L., P.M. and B.O. hold shares in ImmuneWatch BV, an immunoinformatics company., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases, characterized by a treatment-resistant and invasive nature. In line with these inherent aggressive characteristics, only a subset of patients shows a clinical response to the standard of care therapies, thereby highlighting the need for a more personalized treatment approach. In this study, we comprehensively unraveled the intra-patient response heterogeneity and intrinsic aggressive nature of PDAC on bulk and single-organoid resolution. We leveraged a fully characterized PDAC organoid panel (N = 8) and matched our artificial intelligence-driven, live-cell organoid image analysis with retrospective clinical patient response. In line with the clinical outcomes, we identified patient-specific sensitivities to the standard of care therapies (gemcitabine-paclitaxel and FOLFIRINOX) using a growth rate-based and normalized drug response metric. Moreover, the single-organoid analysis was able to detect resistant as well as invasive PDAC organoid clones, which was orchestrates on a patient, therapy, drug, concentration and time-specific level. Furthermore, our in vitro organoid analysis indicated a correlation with the matched patient progression-free survival (PFS) compared to the current, conventional drug response readouts. This work not only provides valuable insights on the response complexity in PDAC, but it also highlights the potential applications (extendable to other tumor types) and clinical translatability of our approach in drug discovery and the emerging era of personalized medicine., (© 2023. The Author(s).)

Background: Whiplash associated disorders (WAD) are the most common non-hospitalised injuries resulting from a motor vehicle crash. Half of individuals with WAD experience ongoing pain and disability. Furthermore, individuals with persistent WAD have lower levels of aerobic capacity and isometric strength compared with age-matched controls. It is not known whether these differences are associated with increased levels of pain and disability, or with reduced physical activity (PA) participation., Objective: Our primary aim was to compare PA levels in individuals with persistent WAD with healthy controls. Secondary aims were to: compare objective and subjective measurements of PA; explore factors that may influence PA; and describe proportions of these populations meeting World Health Organisation PA guidelines., Methods: Objective (ActiGraph accelerometer; seven days) and subjective (International Physical Activity Questionnaire (IPAQ)) PA data were collected for n = 53 age-matched participants (WAD n = 28; controls n = 25)., Results: Independent sample t-tests showed no significant difference in objectively measured PA (p>0.05) between WAD and controls. For the subjective measure (IPAQ), controls reported more overall weekly PA (t = 0.219, p<0.05), while WAD participants reported more weekly walking minutes (t = -0.712, p<0.05). Linear regression showed mental health quality-of-life predicted objectively measured moderate intensity PA (R2 = 0.225, F (2, 44) = 6.379, p<0.004) and subjectively reported overall PA (R2 = 0.132, F (1, 41) = 6.226, p<0.017). Bland-Altman analyses indicated that subjects over-reported MVPA and under-reported sedentary time using the IPAQ., Conclusions: Individuals with WAD had levels of physical and mental health quality-of-life significantly lower than controls and below population norms yet participated in similar levels of PA. Given that increased perceptions of mental health quality-of-life were positively associated with objectively measured MVPA and subjectively reported overall PA, strategies to help people with WAD achieve adequate doses of MVPA may be beneficial. ActiGraph-measured and IPAQ-reported PA were discordant. Hence, IPAQ may not be a reliable measure of habitual PA in WAD., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ritchie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Soft tissue and bone sarcomas are rare entities, hence, standardized therapeutic strategies are difficult to assess. Materials & methods: Immunohistochemistry was performed on 68 sarcoma samples to assess the expression of PD-1, PD-L1, IDO and CD70 in different tumor compartments and molecular analysis was performed to assess microsatellite instability status. Results: PD-1/PD-L1, IDO and CD70 pathways are at play in the immune evasion of sarcomas in general. Soft tissue sarcomas more often show an inflamed phenotype compared with bone sarcomas. Specific histologic sarcoma types show high expression levels of different markers. Finally, this is the first presentation of a microsatellite instability-high Kaposi sarcoma. Discussion/conclusion: Immune evasion occurs in sarcomas. Specific histologic types might benefit from immunotherapy, for which further investigation is needed.

Studying people in their daily life is important for understanding conditions with multi-faceted aetiology such as chronic low back pain. Inertial measurement units can be used to reconstruct the posture and motion of the body outside of laboratories to enable this research. The battery life of these sensors strongly affects the usability of the system, since recharging them frequently is inconvenient and can lead to additional errors. A major determinant of the battery life for these sensors is sampling rate, but the relationship between sampling rate and accuracy in motion reconstruction is not well documented. We measured the spine of 12 participants using inertial measurement units across a variety of tasks such as sitting, standing, walking, and jogging. The orientation of the spine was reconstructed using several filters, including a novel filter developed specifically for high performance at low sampling frequencies. Benchmarking against optical motion capture, we developed a model showing that the error of all tested filters depends exponentially on the sampling frequency, with the optimal filter gains showing a similar exponential relationship. Using this model of error, we developed a criterion for recommending minimum sampling frequencies for accurate motion estimates for each task, finding frequencies ranging from about 13 to 35 Hz sufficient depending on the task. Although we only studied the spine, these models should provide insight into optimizing sampling rate and filter parameters for inertial measurements in general use., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Immunogenic cell death (ICD) refers to an immunologically distinct process of regulated cell death that activates, rather than suppresses, innate and adaptive immune responses. Such responses culminate into T cell-driven immunity against antigens derived from dying cancer cells. The potency of ICD is dependent on the immunogenicity of dying cells as defined by the antigenicity of these cells and their ability to expose immunostimulatory molecules like damage-associated molecular patterns (DAMPs) and cytokines like type I interferons (IFNs). Moreover, it is crucial that the host's immune system can adequately detect the antigenicity and adjuvanticity of these dying cells. Over the years, several well-known chemotherapies have been validated as potent ICD inducers, including (but not limited to) anthracyclines, paclitaxels, and oxaliplatin. Such ICD-inducing chemotherapeutic drugs can serve as important combinatorial partners for anti-cancer immunotherapies against highly immuno-resistant tumors. In this Trial Watch, we describe current trends in the preclinical and clinical integration of ICD-inducing chemotherapy in the existing immuno-oncological paradigms., Competing Interests: No potential conflict of interest was reported by the authors., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Use of the direct oral anticoagulant rivaroxaban has strongly increased in Europe since its market approval for non-valvular atrial fibrillation in 2011. Patients characteristics of rivaroxaban initiators may have changed over time but this has not been investigated so far., Objective: We aimed to describe time trends of patient baseline characteristics among new rivaroxaban users with non-valvular atrial fibrillation from 2011 to 2016/17 in two European countries., Methods: We used data from Germany (German Pharmacoepidemiological Research Database) and the Netherlands (PHARMO Database Network). We included new rivaroxaban users with (i) a first dispensing between 2011 and 2016/17, (ii) ≥ 2 years of age, and (iii) a diagnosis of non-valvular atrial fibrillation and described their baseline medication and comorbidity prior to starting rivaroxaban stratified by year of inclusion., Results: Overall, 130,652 new rivaroxaban users were included during the study period (Germany: N = 127,743, the Netherlands: N = 2909). The sex ratio and median age remained relatively stable over time. The proportion of patients without prior use of oral anticoagulants before initiation of rivaroxaban increased in both countries between 2011 and 2016/17 (Germany: from 51 to 76%, the Netherlands: from 57 to 85%). In Germany, we observed a relative decrease by 27% in the proportion of new rivaroxaban users with a history of ischemic stroke and by 18% in the proportion with a transient ischemic attack at baseline. No such a pattern was observed in the Netherlands. The proportion of patients with heart failure at baseline showed a three-fold increase in the Netherlands, while there was a relative decrease by 12% in Germany., Conclusions: Patient characteristics of new rivaroxaban users with non-valvular atrial fibrillation changed between 2011 and 2016/17, but changes differed between countries. These patterns have methodological implications. They have to be considered in the interpretation of observational studies comparing effectiveness and safety of oral anticoagulants, especially regarding potential bias due to unmeasured confounding., (© 2023. The Author(s).)

Background: CD70-CD27 is a costimulatory ligand-receptor pair in the tumor necrosis factor receptor family. With only limited expression in normal tissues, CD70 is constitutively expressed in a variety of solid tumors and hematologic malignancies, facilitating immunosuppression through CD27 signaling in the tumor microenvironment by enhanced survival of regulatory T cells, induction of T cell apoptosis, and T cell exhaustion. Consequently, CD70 is an increasingly recognized target for developing antibody-based therapies, but its expression patterns vary among different tumor types in spatial distribution, magnitude of expression and percentage of positive cells. In that regard, individual confirmation of CD70 expression at screening and during treatment could enhance the successful implementation of anti-CD70 therapies. Here, we developed a gallium-68 ( 68 Ga) radiolabeled single-domain antibody-fragment targeting CD70 for in vivo positron emission tomography (PET) imaging., Results: An anti-CD70 VHH construct containing a C-direct-tag with a free thiol was developed to enable site-specific conjugation to a NOTA bifunctional chelator for 68 Ga radiolabeling. [ 68 Ga]Ga-NOTA-anti-CD70 VHH was obtained in good radiochemical yield of 30.4 ± 1.7% and high radiochemical purity (> 94%). The radiolabeled VHH showed excellent in vitro and in vivo stability. Specific binding of [ 68 Ga]Ga-NOTA-anti-CD70 VHH was observed on CD70 high 786-O cells, showing significantly higher cell-associated activity when compared to the blocking condition (p < 0.0001) and CD70 low NCl-H1975 cells (p < 0.0001). PET imaging showed specific radiotracer accumulation in CD70 expressing human tumor xenografts, which was efficiently blocked by prior injection of unlabeled anti-CD70 VHH (p = 0.0029). In addition, radiotracer uptake in CD70 high tumors was significantly higher when compared with CD70 low tumors (p < 0.0001). The distribution of the radioactivity in the tumors using autoradiography was spatially matched with immunohistochemistry analysis of CD70 expression., Conclusion: [ 68 Ga]Ga-NOTA-anti-CD70 VHH showed excellent in vivo targeting of CD70 in human cancer xenografts. PET imaging using this radioimmunoconjugate holds promise as a non-invasive method to identify and longitudinally follow-up patients who will benefit most from anti-CD70 therapies., (© 2023. The Author(s).)

Background: A qualitative evaluation study of the prematurely terminated PrEgabalin Lidocaine Capsaicin Neuropathic Pain (PELICAN) study was performed. The PELICAN study aimed to examine pain management for localized neuropathic pain (LNP), as epidemiological figures have shown a high percentage of LNP patients in Belgium. The study compared systemic and topical medications according to pain relief, adverse effects, and several measures of quality of life., Objective: Achieving better study patient recruitment through qualitative research. To investigate and determine the causes of the observed recruitment problems in the PELICAN study, pain centers involved in the study as well as nonrecruiting pain centers were included. Furthermore, it aimed to highlight the positive and negative lessons learned from the conducted study and the number of obstacles the team had to overcome., Methods: A qualitative study, using a mixed methods approach, was performed. Multiple pain centers in Belgium completed an online survey, after which a structured interview was conducted to elaborate the responses in more detail. The broad topics of these meetings were feedback about the study, reviewing survey answers, and actions undertaken to enhance recruitment., Results: Different factors contributed to the low recruitment rate in the PELICAN study, such as limited and late referral from the general practitioners to the Belgian pain centers, insufficient internal referrals from nonpain specialists, lack of specific expertise on LNP in some centers, scarcity of staff, limited reimbursement to administer complex analgesic schemes, overestimation of the patient population, and the reluctance of patients to participate in pain research. Additionally, shortcomings in the implemented study design and the need for more logistical investments were identified., Conclusion: The findings of the qualitative study demonstrate the need for further, more varied LNP research in Belgium, not limited to pharmacological studies. It also sheds important light on the recruitment obstacles that may be faced during these studies. Future studies could support this research by offering better proposals for feasibility and recruitment, for instance, by designing and conducting a compelling pilot study or applying social media during the recruitment phase. Clinical Trials . This trial is registered with NCT03348735. EUDRACT number 2018-003617-17., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Guy H. Hans et al.)

Background: Patient-derived organoids are invaluable for fundamental and translational cancer research and holds great promise for personalized medicine. However, the shortage of available analysis methods, which are often single-time point, severely impede the potential and routine use of organoids for basic research, clinical practise, and pharmaceutical and industrial applications., Methods: Here, we developed a high-throughput compatible and automated live-cell image analysis software that allows for kinetic monitoring of organoids, named Organoid Brightfield Identification-based Therapy Screening (OrBITS), by combining computer vision with a convolutional network machine learning approach. The OrBITS deep learning analysis approach was validated against current standard assays for kinetic imaging and automated analysis of organoids. A drug screen of standard-of-care lung and pancreatic cancer treatments was also performed with the OrBITS platform and compared to the gold standard, CellTiter-Glo 3D assay. Finally, the optimal parameters and drug response metrics were identified to improve patient stratification., Results: OrBITS allowed for the detection and tracking of organoids in routine extracellular matrix domes, advanced Gri3D ® -96 well plates, and high-throughput 384-well microplates, solely based on brightfield imaging. The obtained organoid Count, Mean Area, and Total Area had a strong correlation with the nuclear staining, Hoechst, following pairwise comparison over a broad range of sizes. By incorporating a fluorescent cell death marker, intra-well normalization for organoid death could be achieved, which was tested with a 10-point titration of cisplatin and validated against the current gold standard ATP-assay, CellTiter-Glo 3D. Using this approach with OrBITS, screening of chemotherapeutics and targeted therapies revealed further insight into the mechanistic action of the drugs, a feature not achievable with the CellTiter-Glo 3D assay. Finally, we advise the use of the growth rate-based normalised drug response metric to improve accuracy and consistency of organoid drug response quantification., Conclusion: Our findings validate that OrBITS, as a scalable, automated live-cell image analysis software, would facilitate the use of patient-derived organoids for drug development and therapy screening. The developed wet-lab workflow and software also has broad application potential, from providing a launching point for further brightfield-based assay development to be used for fundamental research, to guiding clinical decisions for personalized medicine., (© 2022. The Author(s).)

As recently highlighted by the SARS-CoV-2 pandemic, viruses have become an increasing burden for health, global economy, and environment. The control of transmission by contact with contaminated materials represents a major challenge, particularly in hospital environments. However, the current disinfection methods in hospital settings suffer from numerous drawbacks. As a result, several medical supplies that cannot be properly disinfected are not reused, leading to severe shortages and increasing amounts of waste, thus prompting the search for alternative solutions. In this work, we report that non-thermal plasma (NTP) can effectively inactivate SARS-CoV-2 from non-porous and porous materials commonly found in healthcare facilities. We demonstrated that 5 min treatment with a dielectric barrier discharge NTP can inactivate 100% of SARS-CoV-2 (Wuhan and Omicron strains) from plastic material. Using porcine respiratory coronavirus (surrogate for SARS-CoV-2) and coxsackievirus B3 (highly resistant non-enveloped virus), we tested the NTP virucidal activity on hospital materials and obtained complete inactivation after 5 and 10 min, respectively. We hypothesize that the produced reactive species and local acidification contribute to the overall virucidal effect of NTP. Our results demonstrate the potential of dielectric barrier discharge NTPs for the rapid, efficient, and low-cost disinfection of healthcare materials., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Despite the recent emergence of immune checkpoint inhibitors, clinical outcomes of metastatic NSCLC patients remain poor, pointing out the unmet need to develop novel therapies to enhance the anti-tumor immune response in NSCLC. In this regard, aberrant expression of the immune checkpoint molecule CD70 has been reported on many cancer types, including NSCLC. In this study, the cytotoxic and immune stimulatory potential of an antibody-based anti-CD70 (aCD70) therapy was explored as single agent and in combination with docetaxel and cisplatin in NSCLC in vitro and in vivo . Anti-CD70 therapy resulted in NK-mediated killing of NSCLC cells and increased production of pro-inflammatory cytokines by NK cells in vitro . The combination of chemotherapy and anti-CD70 therapy further enhanced NSCLC cell killing. Moreover, in vivo findings showed that the sequential treatment of chemo-immunotherapy resulted in a significant improved survival and delayed tumor growth compared to single agents in Lewis Lung carcinoma-bearing mice. The immunogenic potential of the chemotherapeutic regimen was further highlighted by increased numbers of dendritic cells in the tumor-draining lymph nodes in these tumor-bearing mice after treatment. The sequential combination therapy resulted in enhanced intratumoral infiltration of both T and NK cells, as well as an increase in the ratio of CD8+ T cells over Tregs. The superior effect of the sequential combination therapy on survival was further confirmed in a NCI-H1975-bearing humanized IL15-NSG-CD34+ mouse model. These novel preclinical data demonstrate the potential of combining chemotherapy and aCD70 therapy to enhance anti-tumor immune responses in NSCLC patients., Competing Interests: No potential conflict of interest was reported by the authors., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Wilms' tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT1 37-45 -specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT1 37-45 -pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT1 37-45 tetramers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: To gain insights into the underlying mechanisms of NTP therapy sensitivity and resistance, using the first-ever NTP-resistant cell line derived from sensitive melanoma cells (A375)., Methods: Melanoma cells were exposed to NTP and re-cultured for 12 consecutive weeks before evaluation against the parental control cells. Whole transcriptome sequencing analysis was performed to identify differentially expressed genes and enriched molecular pathways. Glucose uptake, extracellular lactate, media acidification, and mitochondrial respiration was analyzed to determine metabolic changes. Cell death inhibitors were used to assess the NTP-induced cell death mechanisms, and apoptosis and ferroptosis was further validated via Annexin V, Caspase 3/7, and lipid peroxidation analysis., Results: Cells continuously exposed to NTP became 10 times more resistant to NTP compared to the parental cell line of the same passage, based on their half-maximal inhibitory concentration (IC50). Sequencing and metabolic analysis indicated that NTP-resistant cells had a preference towards aerobic glycolysis, while cell death analysis revealed that NTP-resistant cells exhibited less apoptosis but were more vulnerable to lipid peroxidation and ferroptosis., Conclusions: A preference towards aerobic glycolysis and ferroptotic cell death are key physiological changes in NTP-resistance cells, which opens new avenues for further, in-depth research into other cancer types., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Safe and effective pharmacological treatment is of paramount importance for treating severe psoriasis. Brodalumab, a monoclonal antibody against interleukin (IL) 17 receptor A, was granted marketing authorisation in the EU in 2017. The European Medicines Agency requested a postauthorisation safety study of brodalumab to address potential safety issues raised during drug development regarding major adverse cardiovascular events, suicidal conduct, cancer and serious infections., Methods and Analysis: BRodalumab Assessment of Hazards: A Multinational Safety is a multicentre observational safety study of brodalumab running from 2017 to 2029 using population-based healthcare databases from Denmark, Sweden, Norway, Netherlands, Germany and three different centres in Italy. A distributed database network approach is used, such that only aggregate data are exchanged between sites.Two types of designs are used: a case-time-control design to study acute effects of transient treatment and a variation of the new user active comparator design to study the effects of transient or chronic treatment. As comparators, inhibitors of TNF-α, inhibitors of IL-12 and IL-23, and other inhibitors of cytokine IL-17A are included.In the self-controlled case-time-control design, the risk of developing the outcome of interest during periods of brodalumab use is compared within individuals to the risk in periods without use.In the active comparator cohort design, new users of brodalumab are identified and matched to new users of active comparators. Potential baseline confounders are adjusted for by using propensity score modelling. For outcomes that potentially require large cumulative exposure, an adapted active comparator design has been developed., Ethics and Dissemination: The study is approved by relevant authorities in Denmark, Norway, Sweden, the Netherlands, Germany and Italy in line with the relevant legislation at each site. Data confidentiality is secured by the distributed network approach. Results will be published in peer-reviewed journals., Trial Registration Number: EUPAS30280., Competing Interests: Competing interests: MR and PBJ report participation in research funded by LEO Pharma A/S (no personal fees). participation in studies funded by Alcon, Almirall, Astellas, AstraZeneca, Boehringer-Ingelheim, Novo Nordisk, Servier, Pfizer, Menarini Pharmaceuticals, and LEO Pharma, all regulator mandated phase IV studies, all with funds paid to the institution (no personal fees). JH has received teaching fees from Atrium. HS reports participation in research funded by LEO Pharma A/S (no personal fees). He has received personal consulting fees from Bristol-Myers-Squibb, Novartis, Roche, Merck and Pfizer outside the submitted work. He has received teaching fees from Atrium. KF and ØK reports participation in research projects funded by Novo Nordisk and LEO Pharma, all regulator mandated phase IV studies, all with funds paid to the institution (no personal fees). ES, EH and RH are employees of the PHARMO Institute for Drug Outcomes Research. This independent research institute performs financially supported studies for government and related healthcare authorities and several pharmaceutical companies. AE has received research funding from Pfizer, Eli Lilly, Novartis, Bristol-Myers Squibb, AbbVie, Janssen Pharmaceuticals, the Danish National Psoriasis Foundation, the Simon Spies Foundation and the Kgl Hofbundtmager Aage Bang Foundation, and honoraria as consultant and/or speaker from AbbVie, Almirall, Leo Pharma, Zuellig Pharma, Galápagos NV, Sun Pharmaceuticals, Samsung Bioepis, Pfizer, Eli Lilly and Company, Novartis, Union Therapeutics, Galderma, Dermavant, UCB, Mylan, Bristol-Myers Squibb, Horizon Therapeutics and Janssen Pharmaceuticals. UK, Alessandro Cesare Rosa, and Valeria Belleudi are employees of the Department of Epidemiology ASL Roma1, Lazio Regional Health Service in Rome, Italy. Our publicly funded department performs epidemiological research and receives grants from public national and international organisations. In case of financial support from private organisations, an ethical code of conduct is signed to guarantee transparency and independence. KG, DH and JR are employees of the Centre for Pharmacoepidemiology of the Karolinska Institutet in Sweden, which receives grants from regulatory authorities, pharmaceutical companies, and contract research organisations for performance of drug safety and drug utilisation studies. UH, WS and SS are working at an independent, non-profit research institute, the Leibniz Institute for Prevention Research and Epidemiology-BIPS. Unrelated to this study, BIPS occasionally conducts studies financed by the pharmaceutical industry. Almost exclusively, these are postauthorisation safety studies (PASS) requested by health authorities. GT declares participation to advisory boards on topics not related to this paper and sponsored by the following pharmaceutical companies in the last two years: Eli Lilly; Sanofi; Amgen; Novo Nordisk; Sobi; Gilead; Celgene; Daikii Sankyo. RG is employed by ARS, a public research centre participating in pharmacoepidemiology studies funded by public or private institutions and compliant with the ENCePP Code of Conduct. The budget of ARS is partially sustained by such studies. Lise Skov Ranch is a full time employee of LEO Pharma A/S., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

A fundamental prerequisite for the efficacy of cancer immunotherapy is the presence of functional, antigen-specific T cells within the tumor. Neoantigen-directed therapy is a promising strategy that aims at targeting the host's immune response against tumor-specific antigens, thereby eradicating cancer cells. Initial forays have been made in clinical environments utilizing vaccines and adoptive cell therapy; however, many challenges lie ahead. We provide an in-depth overview of the current state of the field with an emphasis on in silico neoantigen discovery and the clinical aspects that need to be addressed to unlock the full potential of this therapy., Competing Interests: Declaration of interests L.L., S.L., and B.F. are employees and shareholders at myNEO NV. E.S. is a member of the scientific advisory board of Intra-Vacc. W.v.C. is co-founder and shareholder at myNEO NV. K.T. is a scientific founder of and holds equity in eTheRNA Immunotherapies, and is member of the scientific advisory board of myNEO NV. S.H.v.d.B. is named as inventor on the patent for the use of synthetic long peptides as vaccine as well as on a patent for TEIPP neoantigen-specific treatments. He also serves as a paid member of the strategy board of the vaccine companies: ISA Pharmaceuticals, PCI Biotech, DCprime, and AGLAIA, and also performs contract research on neoantigens for Enara BIO and Frame Pharmaceuticals. P.A.O. reports research funding from and has advised Neon Therapeutics, Bristol-Meyers Squibb, Medrck, CytomX, Pfizer, Novartis, Celldex, Amgen, Array, AstraZeneca/MedImmune, Armo BioSciences, Xencor, Oncorus, and Roche/Genentech. C.B. is co-founder and shareholder at myNEO NV., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The safety and effectiveness of rivaroxaban versus vitamin K antagonists (standard of care [SOC]) for stroke prevention in patients with nonvalvular atrial fibrillation (NVAF) was evaluated in Europe., Research Design and Methods: Observational studies were conducted in the UK, the Netherlands, Germany, and Sweden. Primary safety outcomes were hospitalization for intracranial hemorrhage, gastrointestinal bleeding, or urogenital bleeding among new users of rivaroxaban and SOC with NVAF; outcomes were analyzed using cohort (rivaroxaban or SOC use) and nested case-control designs (current vs nonuse). Statistical analyses comparing rivaroxaban and SOC cohorts were not performed., Results: Overall, 162,919 rivaroxaban users and 177,758 SOC users were identified. In the cohort analysis, incidence ranges for rivaroxaban users were 0.25-0.63 events per 100 person-years for intracranial bleeding, 0.49-1.72 for gastrointestinal bleeding, and 0.27-0.54 for urogenital bleeding. Corresponding ranges for SOC users were 0.30-0.80, 0.30-1.42, and 0.24-0.42, respectively. In the nested case-control analysis, current SOC use generally presented a greater risk of bleeding outcomes than nonuse. Rivaroxaban use (vs nonuse) was associated with a higher risk of gastrointestinal bleeding, but a similar risk of intracranial or urogenital bleeding, in most countries. Ischemic stroke incidence ranged from 0.31 to 1.52 events per 100 person-years for rivaroxaban users., Conclusions: Incidences of intracranial bleeding were generally lower with rivaroxaban than with SOC, whereas incidences of gastrointestinal and urogenital bleeding were generally higher. The safety profile of rivaroxaban for NVAF in routine practice is consistent with findings from randomized controlled trials and other studies.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

Background: A high degree of adherence to direct oral anticoagulants is essential for reducing the risk of ischaemic stroke and systemic embolism in patients with atrial fibrillation, owing to the rapid decline in anticoagulation activity when doses are omitted (i.e. rebound effect)., Objective: We aimed to assess the relationship between non-adherence and non-persistence with direct oral anticoagulants and the incidence of ischaemic stroke in patients with atrial fibrillation., Methods: A nested case-control study was conducted in the Netherlands, Italy and Germany among patients with atrial fibrillation starting direct oral anticoagulants between the drug approval date and the end of database availability. Patients with an ischaemic stroke during the follow-up were selected as cases and compared with matched controls (matched on age ± 5 years, sex, year of cohort entry date and CHA 2 DS 2 -VASc-score at cohort entry date). The cohort entry date was the first dispensing date. Study patients were those aged ≥ 45 years, with ≥ 1 year database history, ≥ 1 year follow-up and at least two direct oral anticoagulant dispensings after the cohort entry date. Adherence and persistence to direct oral anticoagulant treatment were defined as the proportion of days covered ≥ 80% or direct oral anticoagulant continuous use between the cohort entry date and the index date (i.e. date of ischaemic stroke), respectively., Results: In The Netherlands, Italy and Germany, 105 cases and 395 controls, 1580 cases and 6248 controls, and 900 cases and 3570 controls were included, respectively. Odds ratios (ORs) for stroke among current users who were non-adherent compared to adherent users were 0.43 (95% confidence interval [CI] 0.09-1.96) in The Netherlands, 1.11 (95% CI 0.98-1.26) in Italy and 1.21 (95% CI 1.01-1.45) in Germany. The risk of stroke was significantly higher among non-persistent users compared with persistent users in all three databases [OR 1.56 (95% CI 1.00-2.44), OR 1.48 (1.32-1.65) and OR 1.91 (95% CI 1.64-2.22), respectively]. In The Netherlands and Germany, the risk of stroke was higher the longer a patient had stopped using direct oral anticoagulants., Conclusions: Both non-adherence (in Germany) and non-persistence increased the risk of stroke, either using a once-daily or twice-daily regime., (© 2022. The Author(s).)

Gap junctions (GJs), essential structures for cell-cell communication, are made of two hemichannels (commonly called connexons), one on each adjacent cell. Found in almost all cells, GJs play a pivotal role in many physiological and cellular processes, and have even been linked to the progression of diseases, such as cancer. Modulation of GJs is under investigation as a therapeutic strategy to kill tumor cells. Furthermore, GJs have also been studied for their key role in activating anti-cancer immunity and propagating radiation- and oxidative stress-induced cell death to neighboring cells, a process known as the bystander effect. While, gap junction (GJ)-based therapeutic strategies are being developed, one major challenge has been the paradoxical role of GJs in both tumor progression and suppression, based on GJ composition, cancer factors, and tumoral context. Therefore, understanding the mechanisms of action, regulation, and the dual characteristics of GJs in cancer is critical for developing effective therapeutics. In this review, we provide an overview of the current understanding of GJs structure, function, and paradoxical pro- and anti-tumoral role in cancer. We also discuss the treatment strategies to target these GJs properties for anti-cancer responses, via modulation of GJ function., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Baysal, Siozopoulou, Zaryouh, Hermans, Lau, Lambrechts, Fransen, De Pauw, Jacobs, Peeters, Pauwels, Vermorken, Smits, Lardon, De Waele and Wouters.)