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The gut microbiota has emerged as an environmental risk factor that affects thrombotic phenotypes in several cardiovascular diseases. Evidence includes the identification of marker species by sequencing studies of the gut microbiomes of patients with thrombotic disease, the influence of antithrombotic therapies on gut microbial diversity, and preclinical studies in mouse models of thrombosis that have demonstrated the functional effects of the gut microbiota on vascular inflammatory phenotypes and thrombus formation. In addition to impaired gut barrier function promoting low-grade inflammation, gut microbiota-derived metabolites have been shown to act on vascular cell types and promote thrombus formation. Therefore, these meta-organismal pathways that link the metabolic capacities of gut microorganisms with host immune functions have emerged as potential diagnostic markers and novel drug targets. In this Review, we discuss the link between the gut microbiota, its metabolites and thromboembolic diseases., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. Springer Nature Limited.)

Anti-science movements brought more than public distrust in science. Perhaps even more worryingly, these movements are also associated with instances of harassment of-and violence against-scientists. However, virtually nothing is known about individuals likely to harass or harm scientists. Across two pre-registered studies (total N = 749), we investigated the role of worldviews (e.g., political ideology, conspiracy mentality, science cynicism), radicalization risk factors (relative deprivation and threat), and personality traits and how these relate to harassment of scientists (both attitudes and behavior). We found that science cynicism-the perception that scientists are incompetent and corrupt-drives approval of scientists' harassment (attitudes), as well as harmful behavior (e.g., refusing to donate money, not signing a petition). Additionally, perceiving scientists as threatening, as well as dark personality traits (psychopathy and narcissism), contributed to approving scientists' harassment. Overall, the present research takes a first step in identifying predictors of the willingness to harm scientists., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval: Ethical approval was granted by the Institutional Review Board of the Department of Psychology at the University of Amsterdam (FMG-1885 and FMG-3833). Both studies were conducted in accordance with relevant guidelines and regulations of the institution. Informed consent was obtained from all participants., (© 2025. The Author(s).)

There are differing views on how learners' feedback-seeking behaviours (FSB) develop during training. With globalisation has come medical student migration and programme internationalisation. Western-derived educational practices may prove challenging for diverse learner populations. Exploring undergraduate activity using a model of FSB may give insight into how FSB evolves and the influence of situational factors, such as nationality and site of study. Our findings seek to inform medical school processes that support feedback literacy. Using a mixed methods approach, we collected questionnaire and interview data from final-year medical students in Ireland, Bahrain, and Malaysia. A validated questionnaire investigated relationships with FSB and goal orientation, leadership style preference, and perceived costs and benefits. Interviews with the same student population explored their FSB experiences in clinical practice, qualitatively, enriching this data. The data were integrated using the 'following the thread' technique. Three hundred and twenty-five of a total of 514 completed questionnaires and 57 interviews were analysed. Learning goal orientation (LGO), instrumental leadership and supportive leadership related positively to perceived feedback benefits (0.23, 0.2, and 0.31, respectively, p  < 0.05). Perceived feedback benefits are related positively to feedback monitoring and inquiry (0.13 and 0.38, respectively, p  < 0.05). The personal cost of feedback is unsupported in quantitative data, but was a strong theme in interviews, as was feedback avoidance, peer feedback, and unsupportive learning environment. No differences were observed across sub-groups based on gender, study site, or student nationality. Integrated analysis describes FSB: avoiding 'unsafe' feedback ( first, do no harm ) and overcoming barriers ( beat the system ) and goal-centred curation ( shop around ) to optimise benefits. Diverse medical students across three continents undertake FSB with careful navigation, as a valued but risky business, that is highly contextualised. Promoting a constructive FSB is complex. Overcoming outdated theory and practices on the wards remains a challenge to psychologically safe, learner-centred feedback.

Background: The iRefer guidelines provide evidence-based recommendations on imaging and are designed to facilitate appropriate referrals and limit unnecessary radiation exposure. In 2017, a review at this institution assessed the appropriateness of imaging referrals. This study provides an update 5 years later on the referral appropriateness and aims to assess what impact the previous review has had on referral appropriateness., Methods: A retrospective study of 945 referrals across GPs, ED, and inpatients was audited against the iRefer guidelines with costs and cumulative dose estimates calculated for inappropriate referrals considering salaries of those involved, the average time spent performing and reporting radiographs, and the median effective dose values., Results: Results show a decrease in the volume of requests overall with the relative proportion of inappropriate requests rising significantly. Inappropriate requests for abdominal X-rays from GPs decreased from 72 to 37.5%, whereas inappropriate ED referrals increased from 38 to 46% and inappropriate inpatient requests remained static at 30%. The proportion of inappropriate GP requests for spinal radiographs significantly increased for cervical, thoracic, and lumbar spine radiographs, respectively (18 to 66%; 47 to 72%; 53 to 80%; p-value < 0.001). Inappropriate radiographs represent an increased financial and dose-based burden., Conclusion: The volume of radiograph requests reduced after over a 5-year interval; however, the relative proportion of inappropriate requests rose significantly. The iRefer guidelines provide a useful resource to ensure that imaging is used appropriately, effectively, and safely; however, more work is needed to ensure that requests are adhering to these guidelines., Competing Interests: Declarations. Ethical approval and consent to participate: This article does not contain any studies with human or animal participants performed by any of the authors. Conflict of Interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Royal Academy of Medicine in Ireland.)

Robot-assisted minimally invasive esophagectomy (RAMIE) for esophageal carcinoma has emerged as the contemporary alternative to conventional laparoscopic minimally invasive (LMIE), hybrid (HE) and open (OE) surgical approaches. No single study has compared all four approaches with a view to postoperative outcomes. A systematic search of electronic databases was undertaken. A network meta-analysis was performed as per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses-network meta-analysis guidelines. Statistical analysis was performed using R and Shiny. Seven randomised controlled trials (RCTs) with 1063 patients were included. Overall, 32.9% of patients underwent OE (350/1063), 11.0% underwent HE (117/1063), 34.0% of patients underwent LMIE (361/1063), and 22.1% of patients underwent RAMIE (235/1063). OE had the lowest anastomotic leak rate 7.7% (27/350), while LMIE had the lowest pulmonary 10.8% (39/361), cardiac 0.56% (1/177) complications, re-intervention rates 5.08% (12/236), 90-day mortality 1.05% (2/191), and shortest length of hospital stay (mean 11.25 days). RAMIE displayed the lowest 30-day mortality rate at 0.80% (2/250). There was a significant increase in pulmonary complications for those undergoing OE (OR 3.63 [95% confidence interval: 1.4-9.77]) when compared to RAMIE. LMIE is a safe and feasible option for esophagectomy when compared to OE and HE. The upcoming RCTs will provide further data to make a more robust interrogation of the surgical outcomes following RAMIE compared to conventional open surgery to determine equipoise or superiority of each approach as the era of minimally invasive esophagectomy continues to evolve (International Prospective Register of Systematic Reviews Registration: CRD42023438790)., (© The Author(s) 2024. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hookworms are soil-transmitted parasitic nematodes that penetrate the host skin before migrating to the lungs. With an estimated 500-700 million people infected worldwide, hookworm infections are a neglected tropical disease and a significant cause of morbidity, particularly in children, pregnant women, and immunocompromised individuals. Although there is ample evidence that complement activation is pivotal to elicit a protective host immune response against invasive pathogens, its role in hookworm infection remains insufficiently explored. Here, we investigated the complement anaphylatoxin, C5a, during the early lung stage of infection with Nippostrongylus brasiliensis in C57BL/6J wild type and C5aR1 -/- mice. Despite the previously reported ability of lung larvae to evade complement activation, C5a was detectable locally in lung tissue and bronchoalveolar lavages. Surprisingly, C5aR1 presence directly contributed to the pathogenicity of hookworm infection. The burden of viable parasites in the lungs was mitigated in C5aR1 -/- mice, compared to C57BL/6J mice 48 hours post-infection. Additionally, C5aR1 -/- mice showed significantly reduced lung injury, lower cytokine release, attenuated alveolar hemorrhage, and limited alveolar-capillary barrier disruption. Neutrophils were the most abundant and highest C5aR1-expressing cell type in the alveolar space after infection. Deficiency of C5aR1 reduced the influx of neutrophils, monocytes, and eosinophils to the pulmonary airways. RNA sequencing of alveolar neutrophils revealed C5aR1-dependent regulation of the novel nuclear protein, DEDD2. In conclusion, our findings highlight the impact of C5aR1 signaling in neutrophils during hookworm infection uncovering an unexpected downside of complement activation in parasitic infection., 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 © 2024 Walachowski, Garo, Sharma, Jayaraman, Noon, Reinhardt and Bosmann.)

Competing Interests: Competing interests statement:The authors declare no competing interest.

This article explores the emergence of molecular approaches in German genetic research during the 1958-1968 decade as a period of contingency and alternative possibilities. We introduce "Narratives of Contingency" as an analytical framework to examine how scientists construct a specific narrative - linking past experiences with expectations of future conditions - in order to outline and navigate pathway-decisions in the present. We apply this framework to Hans-Jörg Rheinberger's developmental model of molecular genetics and illustrate how the stages he identifies - the direction of the field, institutional developments, and epistemological demarcations - were already central themes in the comparative practices underlying narratives of contingency in this early period. Narratives of contingency can thus serve as a systematic framework for analyzing the processes through which new scientific fields, institutions, and epistemic horizons emerge, and possibly also for identifying historically plausible fork moments or alternative pathways not taken., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Intestinal ischemia-reperfusion (I/R) injury is an acute condition characterized by tissue damage resulting from restricted blood flow to the mesenteric vessels, leading to both local and systemic pathologies with a poor prognosis. Both ischemia and reperfusion trigger a series of cellular and molecular responses, with inflammatory cells serving as key regulators of the pathology. These interactions with the ischemic endothelium are mediated by multiple adhesion receptors. Several animal models have been established to mimic this pathology and investigate the involved molecular pathways. In this study, a microsurgical model of I/R injury is combined with intravital microscopy to visualize leukocyte rolling, adhesion, and neutrophil extracellular trap (NET) formation. This model is applied to transgenic mice deficient in endothelial PAR1 (F2r) to assess the impact of PAR1 on leukocyte rolling and NET formation 1 h after ischemia and immediately following reperfusion. In vivo, Acridine Orange leukocyte staining was employed, and NETs were visualized using a nucleic acid stain. Interestingly, reduced leukocyte adhesion and NET formation were observed in mice lacking the endothelial PAR1 receptor. This model enables the in vivo analysis of key regulators involved in I/R injury.

Background: Atrophy of white and grey matter volumes occurs early in the brains of people with multiple sclerosis (pwMS) and has great clinical relevance. In clinical trials, brain atrophy can be quantified by magnetic resonance imaging (MRI) with automated software tools., Methods: In this study, we analyze volumes of various brain regions with the software "md brain" based on routine MRI scans of 53 pwMS in a real-world setting. We compare brain volumes of pwMS with an EDSS ≥ 3.5 and a disease duration ≥ 10 years to the brain volumes of pwMS with an EDSS < 3.5 and a disease duration < 10 years as well as with or without immunotherapy., Results: pwMS with an EDSS ≥ 3.5 and a disease duration ≥ 10 years had significantly lower volumes of the total brain, the grey matter and of the frontal, temporal, parietal and occipital lobe regions as compared to pwMS with an EDSS < 3.5 and a disease duration < 10 years. Regional brain volumes were significantly lower in pwMS without immunotherapy., Conclusions: The study showed that higher EDSS, longer disease duration and absence of immunotherapy was associated with lower volumes in a number of brain regions. Further real-world studies may include larger patient cohorts in longitudinal analyses., (© 2024. The Author(s).)

Background: Prostate cancer ranks among the most prevalent cancers affecting men globally. While conventional MRI serves as a diagnostic tool, its extended acquisition time, associated costs, and strain on healthcare systems, underscore the necessity for more efficient methods. The emergence of AI-acceleration in prostate MRI offers promise to mitigate these challenges., Methods: A systematic review of studies looking at AI-accelerated prostate MRI was conducted, with a focus on acquisition time along with various qualitative and quantitative measurements., Results: Two primary findings were observed. Firstly, all studies indicated that AI-acceleration in MRI achieved notable reductions in acquisition times without compromising image quality. This efficiency offers potential clinical advantages, including reduced scan durations, improved scheduling, diminished patient discomfort, and economic benefits. Secondly, AI demonstrated a beneficial effect in reducing or maintaining artefact levels in T2-weighted images despite this accelerated acquisition time. Inconsistent results were found in all other domains, which were likely influenced by factors such as heterogeneity in methodologies, variability in AI models, and diverse radiologist profiles. These variances underscore the need for larger, more robust studies, standardization, and diverse training datasets for AI models., Conclusion: The integration of AI-acceleration in prostate MRI thus far shows some promising results for efficient and enhanced scanning. These advancements may fill current gaps in early detection and prognosis. However, careful navigation and collaborative efforts are essential to overcome challenges and maximize the potential of this innovative and evolving field., Advances in Knowledge: This article reveals overall significant reductions in acquisition time without compromised image quality in AI-accelerated prostate MRI, highlighting potential clinical and diagnostic advantages., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Institute of Radiology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Advanced age is associated with an increased susceptibility to Coronavirus Disease (COVID)-19 and more severe outcomes, although the underlying mechanisms are understudied. The lung endothelium is located next to infected epithelial cells and bystander inflammation may contribute to thromboinflammation and COVID-19-associated coagulopathy. Here, we investigated age-associated SARS-CoV-2 pathogenesis and endothelial inflammatory responses using humanized K18-hACE2 mice. Survival was reduced to 20% in aged mice (85-112 weeks) versus 50% in young mice (12-15 weeks) at 10 days post infection (dpi). Bulk RNA-sequencing of endothelial cells from mock and infected mice at 2dpi of both age groups (aged: 72-85 weeks; young: 15 weeks) showed substantially lower significant differentially regulated genes in infected aged mice than in young mice (712 versus 2294 genes). Viral recognition and anti-viral pathways such as RIG-I-like receptor signaling, NOD-like receptor signaling and interferon signaling were regulated in response to SARS-CoV-2. Young mice showed several fold higher interferon responses ( Ifitm3 , Ifit1 , Isg15, Stat1 ) and interferon-induced chemokines ( Cxcl10 and Cxcl11 ) than aged mice. Endothelial cells from infected young mice displayed elevated expression of chemokines ( Cxcl9 , Ccl2 ) and leukocyte adhesion markers ( Icam1 ) underscoring that inflammation of lung endothelium during infection could facilitate leukocyte adhesion and thromboinflammation. TREM1 and acute phase response signaling were particularly prominent in endothelial cells from infected young mice. Immunohistochemistry was unable to detect viral protein in pulmonary endothelium. In conclusion, our data demonstrate that the early host response of the endothelium to SARS-CoV-2 infection declines with aging, which could be a potential contributor to disease severity., Competing Interests: Authors MB and FD were funded by ARCA Biopharma for another project on COVID-19. The remaining 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Subramaniam, Kenney, Jayaraman, O’Connell, Walachowski, Montanaro, Reinhardt, Colucci, Crossland, Douam and Bosmann.)

Objectives and Background: Congenital malformations of the kidney and urinary tract (CAKUT) have a prevalence of 4-60 in 10,000 livebirths and constitute for 40-50% of all end stage pediatric kidney disease. CAKUT can have a genetic background due to monogenetic inherited disease, such as PKD or ciliopathies. They can also be found in combination with extra-renal findings as part of a syndrome. Upon detection of genitourinary malformations during the fetal anomaly scan the question arises if further genetic testing is required. The purpose of this study was to determine the phenotypic presentation of CAKUT cases and the results of exome analysis (WES)., Methods: This is a retrospective analysis of 63 fetal cases with a diagnosis of CAKUT or DSD at a single center between August 2018 and December 2022., Results: A total of 63 cases (5.6%) out of 1123 matched CAKUT phenotypes including renal parenchyma malformations. In 15 out of 63 WES analysis a pathogenic variant was detected (23.8%). In fetuses with isolated CAKUT the rate of detecting a pathogenic variant on exome sequencing was five out of 44 (11.4%). Ten out of 19 fetuses (52.6%) that displayed extra-renal findings in combination with CAKUT were diagnosed with a pathogenic variant., Conclusions: WES provides an increase in diagnosing pathogenic variants in cases of prenatally detected CAKUT. Especially in fetuses with extra-renal malformations, WES facilitates a gain in information on the fetal genotype to enhance prenatal counselling and management., (© 2023. The Author(s).)

Background: Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis., Methods: Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies., Results: Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT 2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V + procoagulant endothelial CD62E + (E-selectin) and neutrophil (Ly6G + ) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082., Conclusions: Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment., Competing Interests: Disclosures None.

In biomedical research, germ-free and gnotobiotic mouse models enable the mechanistic investigation of microbiota-host interactions and their role on (patho)physiology. Throughout any gnotobiotic experiment, standardized and periodic microbiological testing of defined gnotobiotic housing conditions is a key requirement. Here, we review basic principles of germ-free isolator technology, the suitability of various sterilization methods, and the use of sterility testing methods to monitor germ-free mouse colonies. We also discuss their effectiveness and limitations, and share the experience with protocols used in our facility. In addition, possible sources of isolator contamination are discussed and an overview of reported contaminants is provided., 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 © 2023 Dremova, Mimmler, Paeslack, Khuu, Gao, Bosmann, Garo, Schön, Mechler, Beneich, Rebling, Mann, Pontarollo, Kiouptsi and Reinhardt.)

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFβ and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly ( P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls ( n = 10). The pulmonary sources of externalized histones were Ly6G + CD11b + neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFβ1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM + cells to antagonize macrophage-derived IL-27 production. TGFβ1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFβ1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.

Germline pathogenic variants in isocitrate dehydrogenase 1 (IDH1) can lead to a rare neurodevelopmental disorder called metaphyseal chondromatosis with D-2-hydroxyglutaric aciduria, including severe skeletal and cerebral anomalies. To the best of our knowledge, no prenatal case of an IDH1 pathogenic variant has been reported in literature. Somatic sequence variants in IDH1/2 genes are described in distinct cancers, premalignant diseases and rare inherited metabolic disorders. Amniocentesis and further genetic testing including trio exome sequencing were performed due to suspicious findings on a second trimester routine prenatal ultrasound examination. The fetus was found to have growth restriction, cerebral abnormalities (ex vacuo hydrocephalus, cerebellar and vermian hypoplasia, corpus callosum dysgenesis), brachycephaly, narrow chest, persistent left superior vena cava, liver calcifications, hyperechogenic bowel, short tubular bones and joint contractures. A de novo heterozygous variant in the IDH1 gene was detected via trio exome sequencing. The prenatal diagnosis of a de novo pathogenic variant in IDH1 in a fetus with the described phenotype, obtained through trio exome sequencing, helped parents and providers with an informed decision making about pregnancy management., (© 2023 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

STANDARDIZED IN VITRO AND IN VIVO MODEL SYSTEMS TO SIMPLIFY COMPLEXITY-THAT'S HOW WE LEARN: The discovery of new target molecules and translational progress in the development and refinement of antithrombotic therapies as well as the improved treatment of bleeding disorders strongly relies on standardized ex vivo and in vivo models that closely resemble the respective human pathologies. The standardization of these models requires sound training in specialized hemostasis and thrombosis research laboratories as well as a consistent daily routine. In this theme issue of Hämostaseologie-Progress in Haemostasis , four review articles cover key models that have proven instrumental to gain mechanistic insights on thrombogenesis and hemostatic processes. In recent decades, these models have moved our field forward and enabled translation across scales, from cell-based research to isolated flow chamber systems, to mouse thrombosis models reflecting the pathologic situations as observed in patients, to large animal models., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Thieme. All rights reserved.)

The gut microbiota is increasingly recognized as an actuating variable shaping vascular development and endothelial cell function in the intestinal mucosa but also affecting the microvasculature of remote organs. In the small intestine, colonization with gut microbiota and subsequent activation of innate immune pathways promotes the development of intricate capillary networks and lacteals, influencing the integrity of the gut-vascular barrier as well as nutrient uptake. Since the liver yields most of its blood supply via the portal circulation, the hepatic microcirculation steadily encounters microbiota-derived patterns and active signaling metabolites that induce changes in the organization of the liver sinusoidal endothelium, influencing immune zonation of sinusoids and impacting on metabolic processes. In addition, microbiota-derived signals may affect the vasculature of distant organ systems such as the brain and the eye microvasculature. In recent years, this gut-resident microbial ecosystem was revealed to contribute to the development of several vascular disease phenotypes., (Copyright © 2023 Cold Spring Harbor Laboratory Press; all rights reserved.)

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i.e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID-19-associated coagulopathy is revisited., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/).)

IMA101 is an actively personalized, multi-targeted adoptive cell therapy (ACT), whereby autologous T cells are directed against multiple novel defined peptide-HLA (pHLA) cancer targets. HLA-A*02:01-positive patients with relapsed/refractory solid tumors expressing ≥1 of 8 predefined targets underwent leukapheresis. Endogenous T cells specific for up to 4 targets were primed and expanded in vitro. Patients received lymphodepletion (fludarabine, cyclophosphamide), followed by T-cell infusion and low-dose IL2 (Cohort 1). Patients in Cohort 2 received atezolizumab for up to 1 year (NCT02876510). Overall, 214 patients were screened, 15 received lymphodepletion (13 women, 2 men; median age, 44 years), and 14 were treated with T-cell products. IMA101 treatment was feasible and well tolerated. The most common adverse events were cytokine release syndrome (Grade 1, n = 6; Grade 2, n = 4) and expected cytopenias. No patient died during the first 100 days after T-cell therapy. No neurotoxicity was observed. No objective responses were noted. Prolonged disease stabilization was noted in three patients lasting for 13.7, 12.9, and 7.3 months. High frequencies of target-specific T cells (up to 78.7% of CD8+ cells) were detected in the blood of treated patients, persisted for >1 year, and were detectable in posttreatment tumor tissue. Individual T-cell receptors (TCR) contained in T-cell products exhibited broad variation in TCR avidity, with the majority being low avidity. High-avidity TCRs were identified in some patients' products. This study demonstrates the feasibility and tolerability of an actively personalized ACT directed to multiple defined pHLA cancer targets. Results warrant further evaluation of multi-target ACT approaches using potent high-avidity TCRs. See related Spotlight by Uslu and June, p. 865., (©2023 American Association for Cancer Research.)

Keratinocytes (KCs) form the outer epithelial barrier of the body, protecting against invading pathogens. Mice lacking the IL-17RA or both IL-17A and IL-17F develop spontaneous Staphylococcusaureus skin infections. We found a marked expansion of T 17 cells, comprised of RORγt-expressing γδ T cells and T helper 17 cells in the skin-draining lymph nodes of these mice. Contradictory to previous suggestions, this expansion was not a result of a direct negative feedback loop because we found no expansion of T 17 cells in mice lacking IL-17 signaling specifically in T cells. Instead, we found that the T 17 expansion depended on the microbiota and was observed only when KCs were deficient for IL-17RA signaling. Indeed, mice that lack IL-17RA only in KCs showed an increased susceptibility to experimental epicutaneous infection with S. aureus together with an accumulation of IL-17A-producing γδ T cells. We conclude that deficiency of IL-17RA on KCs leads to microbiota dysbiosis in the skin, which triggers the expansion of IL-17A-producing T cells. Our data show that KCs are the primary target cells of IL-17A and IL-17F, coordinating the defense against microbial invaders in the skin., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1 ΔIEC ) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1 ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling., (© 2023. The Author(s).)

Polyphosphates are highly conserved, linear polymers of monophosphates that reside in all living cells. Bacteria produce long chains containing hundreds to thousands of phosphate units, which can interfere with host defense to infection. Here, we report that intratracheal long-chain polyphosphate administration to C57BL/6J mice resulted in the release of proinflammatory cytokines and influx of Ly6G + polymorphonuclear neutrophils in the bronchoalveolar lavage fluid causing a disruption of the physiologic endothelial-epithelial small airway barrier and histologic signs of lung injury. Polyphosphate-induced effects were attenuated after neutrophil depletion in mice. In isolated murine neutrophils, long-chain polyphosphates modulated cytokine release induced by lipopolysaccharides (LPS) from Gram-negative bacteria or lipoteichoic acid from Gram-positive bacteria. In addition, long-chain polyphosphates induced immune evasive effects in human neutrophils. In detail, long-chain polyphosphates downregulated CD11b and curtailed the phagocytosis of Escherichia coli particles by neutrophils. Polyphosphates modulated the migration capacity by inducing CD62L shedding resulting in CD62L low and CD11b low neutrophils. The release of IL-8 induced by LPS was also significantly reduced. Pharmacologic blockade of PI3K with wortmannin antagonized long-chain polyphosphate-induced effects on LPS-induced IL-8 release. In conclusion, polyphosphates govern immunomodulation in murine and human neutrophils, suggesting polyphosphates as a therapeutic target for bacterial infections to restore innate immune defense., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Aging is associated with cognitive decline and memory loss in humans. In rats, aging-associated neuronal excitability changes and impairments in learning have been extensively studied in the hippocampus. Here, we investigated the roles of L-type calcium channels (LTCCs) in the rat piriform cortex (PC), in comparison with those of the hippocampus. We employed spatial and olfactory tasks that involve the hippocampus and PC. LTCC blocker nimodipine administration impaired spontaneous location recognition in adult rats (6-9 months). However, the same blocker rescued the spatial learning deficiency in aged rats (19-23 months). In an odor-associative learning task, infusions of nimodipine into either the PC or dorsal CA1 impaired the ability of adult rats to learn a positive odor association. Again, in contrast, nimodipine rescued odor associative learning in aged rats. Aged CA1 neurons had higher somatic expression of LTCC Cav1.2 subunits, exhibited larger afterhyperpolarization (AHP) and lower excitability compared with adult neurons. In contrast, PC neurons from aged rats showed higher excitability and no difference in AHP. Cav1.2 expression was similar in adult and aged PC somata, but relatively higher in PSD95- puncta in aged dendrites. Our data suggest unique features of aging-associated changes in LTCCs in the PC and hippocampus., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Cells of the innate immune system, including monocytes and neutrophils, are key players in the process of venous thrombosis. T lymphocytes have recently been implicated in venous thrombus resolution but the role of B lymphocytes in thrombosis is unknown. The present study was conducted to address this question using a mouse model of partial ligation of the inferior vena cava. Although only a very low number of B cells was found in the venous thrombi of wild-type mice, B cell-deficient JHT mutant mice developed larger venous thrombi than the wild-type controls. Consistent with enhanced thrombogenesis, increased neutrophil counts were found in the circulating blood and in the thrombi of B cell-deficient mice. One of the mechanisms by which neutrophils contribute to venous thrombosis is the formation of neutrophil extracellular traps (NETs). In agreement, higher quantities of NETs were observed in the thrombi of B cell-deficient mice. In vitro assays showed no difference in the NET building capacity of the isolated neutrophils between B cell-deficient and wild-type mice, indicating that the enhanced NET formation in the thrombi of B cell-deficient mice is attributable to the increased number of circulating neutrophils in these animals. Furthermore, increased concentration of the clot-stabilizing macromolecule fibrinogen was detected in the plasma of B cell-deficient mice. In conclusion, B cell-deficiency in mice indirectly promotes venous thrombosis by increasing neutrophil numbers and elevating fibrinogen levels., Competing Interests: The authors declare no conflict of interest. S.K. received consultancy honoraria from Almiral not associated with this work., (© 2022 The Author(s).)

Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G + polymorphonuclear neutrophils, depletion of CD11c + SiglecF + alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1 -/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury., 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 Roewe, Walachowski, Sharma, Berthiaume, Reinhardt and Bosmann.)

Human TRIAP1 (TP53-regulated inhibitor of apoptosis 1; also known as p53CSV for p53-inducible cell survival factor) is the homolog of yeast Mdm35, a well-known chaperone that interacts with the Ups/PRELI family proteins and participates in the intramitochondrial transfer of lipids for the synthesis of cardiolipin (CL) and phosphatidylethanolamine. Although recent reports indicate that TRIAP1 is a prosurvival factor abnormally overexpressed in various types of cancer, knowledge about its molecular and metabolic function in human cells is still elusive. It is therefore critical to understand the metabolic and proliferative advantages that TRIAP1 expression provides to cancer cells. Here, in a colorectal cancer cell model, we report that the expression of TRIAP1 supports cancer cell proliferation and tumorigenesis. Depletion of TRIAP1 perturbed the mitochondrial ultrastructure, without a major impact on CL levels and mitochondrial activity. TRIAP1 depletion caused extramitochondrial perturbations resulting in changes in the endoplasmic reticulum-dependent lipid homeostasis and induction of a p53-mediated stress response. Furthermore, we observed that TRIAP1 depletion conferred a robust p53-mediated resistance to the metabolic stress caused by glutamine deprivation. These findings highlight the importance of TRIAP1 in tumorigenesis and indicate that the loss of TRIAP1 has extramitochondrial consequences that could impact on the metabolic plasticity of cancer cells and their response to conditions of nutrient deprivation., 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 Nedara, Reinhardt, Lebraud, Arena, Gracia, Buard, Pioche-Durieu, Castelli, Colsch, Bénit, Rustin, Albaud, Gestraud, Baulande, Servant, Deutsch, Verbavatz, Brenner, Milliat and Modjtahedi.)

Treatment success of head and neck squamous cell carcinoma (HNSCC) is often hindered by cisplatin resistance. As inherent and acquired therapy resistance counteracts improvement in long-term survival, novel multi-targeting strategies triggering cancer cell apoptosis are urgently required. Here, we identify the vitamin D receptor (VDR) as being significantly overexpressed in tumors of HNSCC patients (n = 604; p = 0.0059), correlating with tumor differentiation ( p = 0.0002), HPV status ( p = 0.00026), and perineural invasion ( p = 0.0087). The VDR, a member of the nuclear receptor superfamily, is activated by its ligand vitamin D (VitD) and analogs, triggering multiple cellular responses. As we found that the VDR was also upregulated in our cisplatin-resistant HNSCC models, we investigated its effect on overcoming cisplatin resistance. We discovered that VitD/cisplatin combinations synergistically killed even cisplatin-resistant cells at clinically achievable levels. Similar results were obtained for the clinically used VitD analog Maxacalcitol. Moreover, VitD/cisplatin combinations inhibited tumor cell migration by E-cadherin upregulation. Signaling pathway analyses revealed that VitD co-treatments triggered cancer cell death by increasing the expression of the pro-apoptotic BCL-2 family protein BIM. BIM's pro-apoptotic activity in HNSCC cells was confirmed by ectopic overexpression studies. Importantly, BIM expression is positively associated with HNSCC patients' (n = 539) prognosis, as high expression correlated with improved survival ( p = 0.0111), improved therapy response ( p = 0.0026), and remission ( p = 0.004). Collectively, by identifying, for the first time, the VDR/BIM axis, we here provide a molecular rationale for the reported anti-cancer activity of VitD/analogs in combination therapies. Our data also suggest its exploitation as a potential strategy to overcome cisplatin resistance in HNSCC and other malignancies by inducing additional pro-apoptotic pathways.

The essential amino acid tryptophan (Trp) is metabolized by gut commensals, yielding in compounds that affect innate immune cell functions directly, but also acting on the aryl hydrocarbon receptor (AHR), thus regulating the maintenance of group 3 innate lymphoid cells (ILCs), promoting T helper 17 (T H 17) cell differentiation, and interleukin-22 production. In addition, microbiota-derived Trp metabolites have direct effects on the vascular endothelium, thus influencing the development of vascular inflammatory phenotypes. Indoxyl sulfate was demonstrated to promote vascular inflammation, whereas indole-3-propionic acid and indole-3-aldehyde had protective roles. Furthermore, there is increasing evidence for a contributory role of microbiota-derived indole-derivatives in blood pressure regulation and hypertension. Interestingly, there are indications for a role of the kynurenine pathway in atherosclerotic lesion development. Here, we provide an overview on the emerging role of gut commensals in the modulation of Trp metabolism and its influence in cardiovascular disease development., (© 2022. The Author(s).)

Rapid and reliable fungal identification is crucial to delineate infectious diseases, and to establish appropriate treatment for onychomycosis. Compared to conventional diagnostic methods, molecular techniques are faster and feature higher accuracy in fungal identification. However, in current clinical practice, molecular mycology is not widely available, and its practical applicability is still under discussion. This study summarizes the results of 16,094 consecutive nail specimens with clinical suspicion of onychomycosis. We performed PCR/sequencing on all primary nail specimens for which conventional mycological diagnostics remained inconclusive. In specimens with a positive direct microscopy but negative or contaminated culture, molecular mycology proved superior and specified a fungal agent in 65% (587/898). In 75% (443/587), the identified pathogen was a dermatophyte. Positive cultures for dermatophytes, yeasts and non-dermatophyte molds (NDMs) were concordant with primary-specimen-DNA PCR/sequencing in 83% (10/12), 34% (22/65) and 45% (76/169), respectively. Among NDMs, agreement was high for Fusarium spp. (32/40; 80%), but low for Penicillium spp. (5/25; 20%) and Alternaria spp. (1/20; 5%). This study underlines the improvement in diagnostic yield by fungal primary-specimen-DNA PCR/sequencing in the event of a negative or contaminated culture, as well as its significance for the diagnosis of dermatophyte and non-dermatophyte onychomycosis. Molecular mycology methods like PCR and DNA sequencing should complement conventional diagnostics in cases of equivocal findings, suspected NDM onychomycosis or treatment-resistant nail pathologies.

T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 ( COL6A3 ) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.

Purpose: Smoking cessation in patients with diagnosed lung cancer has positive effects on cancer therapy and overall prognosis. Despite this, knowledge on smoking cessation in lung cancer patients is sparse., Methods: This is an observational single centre, 12-week, prospective, single-arm trial at a tertiary lung cancer centre. Responsive patients were enrolled following confirmed lung cancer diagnosis. Smoking cessation intervention included counselling as well as pharmacotherapy. The primary endpoint was the point prevalence abstinence rate at week 12 based on biochemical verification. Secondary endpoints were the abstinence rate at week 26, quality of life and side effects., Results: 80 patients were enrolled. Mean age was 62.6 ± 7.9 years. Most patients (63%) were treated with chemotherapy or radiochemotherapy. 39 patients used nicotine replacement therapy, 35 varenicline whereas six patients did not use pharmacotherapy. During the study period 13 patients died. Data were available in 72 patients after 12 weeks and 57 patients at week 24. Point prevalence abstinence rates were 37.5% (95% CI 26.4-49.7%) at week 12 and 32.8% (95% CI 21.8-45.4%) at week 26, respectively. Quality of life and side effects were not significantly affected by pharmacotherapy., Conclusion: In conclusion, our results suggest that smoking cessation is feasible in patients with newly diagnosed lung cancer. The observed abstinence rate is comparable to other patient cohorts. Furthermore, pharmacotherapy in addition to cancer therapy was safe and did not show novel side effects in these seriously ill patients. Thus, smoking cessation should be an integral part of lung cancer treatment. Trial registration The study was conducted in accordance with good clinical practice standards (GCP) and approved by the local ethics committee (16/3/14), the European PAS registry (EUPAS8748) and the German BfArM (NIS-Studien-Nr. 5508). All patients provided written informed consent before study enrollment., (© 2022. The Author(s).)

Purpose: Immunotherapy for hepatocellular carcinoma (HCC) shows considerable promise in improving clinical outcomes. HepaVac-101 represents a single-arm, first-in-human phase I/II multicenter cancer vaccine trial for HCC (NCT03203005). It combines multipeptide antigens (IMA970A) with the TLR7/8/RIG I agonist CV8102. IMA970A includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides selected after mass spectrometric identification in human HCC tissues or cell lines. CV8102 is an RNA-based immunostimulator inducing a balanced Th1/Th2 immune response., Patients and Methods: A total of 82 patients with very early- to intermediate-stage HCCs were enrolled and screened for suitable HLA haplotypes and 22 put on study treatment. This consisted in a single infusion of low-dose cyclophosphamide followed by nine intradermal coadministrations of IMA970A and CV8102. Only patients with no disease relapse after standard-of-care treatments were vaccinated. The primary endpoints of the HepaVac-101 clinical trial were safety, tolerability, and antigen-specific T-cell responses. Secondary or exploratory endpoints included additional immunologic parameters and survival endpoints., Results: The vaccination showed a good safety profile. Transient mild-to-moderate injection-site reactions were the most frequent IMA970A/CV8102-related side effects. Immune responses against ≥1 vaccinated HLA class I tumor-associated peptide (TAA) and ≥1 vaccinated HLA class II TAA were respectively induced in 37% and 53% of the vaccinees., Conclusions: Immunotherapy may provide a great improvement in treatment options for HCC. HepaVac-101 is a first-in-human clinical vaccine trial with multiple novel HLA class I- and class II-restricted TAAs against HCC. The results are initial evidence for the safety and immunogenicity of the vaccine. Further clinical evaluations are warranted., (©2022 American Association for Cancer Research.)

The biocompatibility of carrier nanomaterials in blood is largely hampered by their activating or inhibiting role on the clotting system, which in many cases prevents safe intravascular application. Here, we characterized an aqueous colloidal ethyl hydroxyethyl cellulose (EHEC) solution and tested its effect on ex vivo clot formation, platelet aggregation, and activation by thromboelastometry, aggregometry, and flow cytometry. We compared the impact of EHEC solution on platelet aggregation with biocompatible materials used in transfusion medicine (the plasma expanders gelatin polysuccinate and hydroxyethyl starch). We demonstrate that the EHEC solution, in contrast to commercial products exhibiting Newtonian flow behavior, resembles the shear-thinning behavior of human blood. Similar to established nanomaterials that are considered biocompatible when added to blood, the EHEC exposure of resting platelets in platelet-rich plasma does not enhance tissue thromboplastin- or ellagic acid-induced blood clotting, or platelet aggregation or activation, as measured by integrin α IIb β 3 activation and P-selectin exposure. Furthermore, the addition of EHEC solution to adenosine diphosphate (ADP)-stimulated platelet-rich plasma does not affect the platelet aggregation induced by this agonist. Overall, our results suggest that EHEC may be suitable as a biocompatible carrier material in blood circulation and for applications in flow-dependent diagnostics.

Polyvinyl polymers bearing pendant hole transport functionalities have been extensively explored for solution-processed hole transport layer (HTL) technologies, yet there are only rare examples of high anisotropic packing of the HT moieties of these polymers into substrate-parallel orientations within HTL films. For small molecules, substrate-parallel alignment of HT moieties is a well-established approach to improve overall device performance. To address the longstanding challenge of extension from vapor-deposited small molecules to solution-processable polymer systems, a fundamental chemistry tactic is reported here, involving the positioning of HT side chains within macromolecular frameworks by the construction of HT polymers having bottlebrush topologies. Applying state-of-the-art polymer synthetic techniques, various functional subunits, including triphenylamine (TPA) for hole transport and adhesion to the substrate, and perfluoro alkyl-substituted benzyloxy styrene for migration to the air interface, were organized with exquisite control over the composition and placement throughout the bottlebrush topology. Upon assembling the HT bottlebrush (HTB) polymers into monolayered HTL films on various substrates through spin-casting and thermal annealing, the backbones of HTBs were vertically aligned while the grafts with pendant TPAs were extended parallel to the substrate. The overall design realized high TPA π-stacking along the out-of-plane direction of the substrate in the HTLs, which doubled the efficiency of organic light-emitting diodes compared with linear poly(vinyl triphenylamine)s.

To understand functional duality of the complement system in host defense and lung injury, a more comprehensive view of its localized production in the lung, and the impact of age on complement production are essential. Here, we explored the expression of complement genes through computational analysis of preexisting single cell RNA sequencing data from lung transcriptomes of healthy young (3 months) and old C57BL/6 mice (24 months), and humans. We characterized the distribution of 48 complement genes. Across 28 distinct immune and non-immune cell types in mice, mesothelial cells expressed the greatest number of complement genes (e.g., C1ra, C2, C3), and regulators (e.g., Serping1, Cfh). C5 was abundant in type II alveolar epithelial cells and C1q in interstitial lung macrophages. There were only moderate differences in gene expression between young and old mice. Among 57 human lung cell types, mesothelial cells showed abundant complement expression. A few differences in gene  expression (e.g., FCN1, CFI, C6, C7) were also evident between mice and human lung cells. Our findings present a novel perspective on the expression patterns of complement genes in normal lungs. These findings highlight the potential functions of complement in tissue-specific homeostasis and immunity and may foster a mechanistic understanding of its role in lung health and disease., (© 2022. The Author(s), under exclusive licence to Society for Mucosal Immunology.)

IL-27 is a heterodimeric IL-12 family cytokine formed by noncovalent association of the promiscuous EBI3 subunit and selective p28 subunit. IL-27 is produced by mononuclear phagocytes and unfolds pleiotropic immune-modulatory functions through ligation to IL-27 receptor α (IL-27RA). Although IL-27 is known to contribute to immunity and to limit inflammation after various infections, its relevance for host defense against multicellular parasites is still poorly defined. Here, we investigated the role of IL-27 during infection with the soil-transmitted hookworm, Nippostrongylus brasiliensis , in its early host intrapulmonary life cycle. IL-27(p28) was detectable in bronchoalveolar lavage fluid of C57BL/6J wild-type mice on day 1 after s.c. inoculation. IL-27RA expression was most abundant on lung-invading γδ T cells. Il27ra -/- mice showed increased lung parasite burden together with aggravated pulmonary hemorrhage and higher alveolar total protein leakage as a surrogate for epithelial-vascular barrier disruption. Conversely, injections of recombinant mouse (rm)IL-27 into wild-type mice reduced lung injury and parasite burden. In multiplex screens, higher airway accumulations of IL-6, TNF-α, and MCP-3 (CCL7) were observed in Il27ra -/- mice, whereas rmIL-27 treatment showed a reciprocal effect. Importantly, γδ T cell numbers in airways were enhanced by endogenous or administered IL-27. Further analysis revealed a direct antihelminthic function of IL-27 on γδ T cells as adoptive intratracheal transfer of rmIL-27-treated γδ T cells during primary N. brasiliensis lung infection conferred protection in mice. In summary, this report demonstrates protective functions of IL-27 to control the early lung larval stage of hookworm infection., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Purpose: To assess the impact of a pharmacist-led warfarin to DOAC conversion initiative during the COVID-19 pandemic. Methods: Patients who were prescribed warfarin and followed with the anticoagulation clinic for INR monitoring were assessed by outpatient clinical pharmacists as potential candidates for transition to DOACs from March-August 2020. Results: 530 patients were assessed for transition to DOACs, of which 373 (70.4%) were deemed by clinical pharmacists to be candidates for DOACs. Of the patients who were candidates for DOACs, 66 (17.7%) were transitioned from warfarin to a DOAC. Of the patients who transitioned to a DOAC, 59 (89.4%) remained on a DOAC after one year. Conclusion: Outpatient clinical pharmacists are an effective resource to help identify patients who are candidates for DOACs and assist with transition from warfarin. Further, high persistence rates with DOAC therapy after one year demonstrate the positive impact of the clinical pharmacist on medication adherence., (© Individual authors.)

The prevalence and clinical importance of arterial hypertension are still growing. Inorganic nitrite (NO 2 - ) represents an attractive dietary antihypertensive agent, but its metabolism and mode of action, which we aimed to investigate with the present study, are not completely understood. Isolated aortic rings from rats were treated ex vivo with oxidants, and rats were infused in vivo with angiotensin-II. Vascular responses to acetylcholine (ACh) and nitrite were assessed by isometric tension recording. The loss of vasodilatory potency in response to oxidants was much more pronounced for ACh as compared to nitrite ex vivo (but not in vivo with angiotensin-II). This effect may be caused by the redox regulation of conversion to xanthine oxidase (XO). Conventionally raised and germ-free mice were treated with nitrite by gavage, which did not improve ACh-mediated vasodilation, but did increase the plasma levels of S-nitros(yl)ated proteins in the conventionally-raised, but not in the germ-free mice. In conclusion, inorganic nitrite represents a dietary drug option to treat arterial hypertension in addition to already established pharmacological treatment. Short-term oxidative stress did not impair the vasodilatory properties of nitrite, which may be beneficial in cardiovascular disease patients. The gastrointestinal microbiome appears to play a key role in nitrite metabolism and bioactivation.

Background: International studies show that dizziness and vertigo are a significant burden on the general population, with 20-30% experiencing symptoms over a lifetime. There are no Irish studies indicating prevalence. The aim of this study was to review primary care referrals for patients with dizziness and vertigo to an otolaryngology tertiary centre., Methods: A review of an out-patient department waiting list was performed on primary care referrals for dizziness and vertigo to an otolaryngology tertiary centre. Demographic information was recorded on all patients referred between May 2017 and August 2019., Results: Two hundred fifteen patients out of 901 patients (24%) referred to an otologist between May 2017 and August 2019 were referred with dizziness as a presenting complaint. The average age was 51 years. F/M ratio was 3:2. The average waiting time was 441 days. The most common associated otological symptom was tinnitus (42%). Relevant comorbidities included anxiety, depression, migraine/headaches and cardiac disease., Conclusion: This study demonstrates that a significant number of patients referred to an otologist from primary care are referred with dizziness and vertigo and supports the need for the establishment of multi-disciplinary vestibular/balance centres to address and manage these patients., (© 2021. Royal Academy of Medicine in Ireland.)

The earliest abnormality associated with Alzheimer's disease (AD) is the presence of persistently phosphorylated pretangle tau in locus coeruleus (LC) neurons. LC neuron numbers and fiber density are positive predictors of cognition prior to death. Using an animal model of LC pretangle tau, we ask if LC activity patterns influence the sequelae of pretangle tau. We seeded LC neurons with a pretangle human tau gene. We provided daily novelty- or stress-associated optogenetic activation patterns to LC neurons for 6 weeks in mid-adulthood and, subsequently, probed cognitive and anatomical changes. Prior LC phasic stimulation prevented spatial and olfactory discrimination deficits and preserved LC axonal density. A stress-associated activation pattern increased indices of anxiety and depression, did not improve cognition, and worsened LC neuronal health. These results argue that variations in environmental experiences associated with differing LC activity patterns may account for individual susceptibility to development of AD in humans., Competing Interests: Qi Yuan has been appointed as a member of the Natural Sciences and Engineering Research Council of Canada (NSERC) Genes, Cells and Molecules Evaluation Group (Neuroscience) for a 3‐year term ending on the June 30, 2021. NSERC funds non–health‐related science and engineering research grants. This work was supported by Canadian Institute of Health Research project grant (#PJT‐16124) to Qi Yuan, received in April 2019 (5 years) and paid to Memorial University. All other authors have nothing to disclose., (© 2021 The Authors. Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring published by Wiley Periodicals, LLC on behalf of Alzheimer's Association.)

Therapy resistance is the major cause of cancer death. As patients respond heterogeneously, precision/personalized medicine needs to be considered, including the application of nanoparticles (NPs). The success of therapeutic NPs requires to first identify clinically relevant resistance mechanisms and to define key players, followed by a rational design of biocompatible NPs capable to target resistance. Consequently, we employed a tiered experimental pipeline from in silico to analytical and in vitro to overcome cisplatin resistance. First, we generated cisplatin-resistant cancer cells and used next-generation sequencing together with CRISPR/Cas9 knockout technology to identify the ion channel LRRC8A as a critical component for cisplatin resistance. LRRC8A's cisplatin-specificity was verified by testing free as well as nanoformulated paclitaxel or doxorubicin. The clinical relevance of LRRC8A was demonstrated by its differential expression in a cohort of 500 head and neck cancer patients, correlating with patient survival under cisplatin therapy. To overcome LRRC8A-mediated cisplatin resistance, we constructed cisplatin-loaded, polysarcosine-based core cross-linked polymeric NPs (NP Cis , Ø ∼ 28 nm) with good colloidal stability, biocompatibility (low immunogenicity, low toxicity, prolonged in vivo circulation, no complement activation, no plasma protein aggregation), and low corona formation properties. 2D/3D-spheroid cell models were employed to demonstrate that, in contrast to standard of care cisplatin, NP Cis significantly ( p < 0.001) eradicated all cisplatin-resistant cells by circumventing the LRRC8A-transport pathway via the endocytic delivery route. We here identified LRRC8A as critical for cisplatin resistance and suggest LRRC8A-guided patient stratification for ongoing or prospective clinical studies assessing therapy resistance to nanoscale platinum drug nanoformulations versus current standard of care formulations.

The gut microbiota affects remote organ functions but its impact on organotypic endothelial cell (EC) transcriptomes remains unexplored. The liver endothelium encounters microbiota-derived signals and metabolites via the portal circulation. To pinpoint how gut commensals affect the hepatic sinusoidal endothelium, a magnetic cell sorting protocol, combined with fluorescence-activated cell sorting, was used to isolate hepatic sinusoidal ECs from germ-free (GF) and conventionally raised (CONV-R) mice for transcriptome analysis by RNA sequencing. This resulted in a comprehensive map of microbiota-regulated hepatic EC-specific transcriptome profiles. Gene Ontology analysis revealed that several functional processes in the hepatic endothelium were affected. The absence of microbiota influenced the expression of genes involved in cholesterol flux and angiogenesis. Specifically, genes functioning in hepatic endothelial sphingosine metabolism and the sphingosine-1-phosphate pathway showed drastically increased expression in the GF state. Our analyses reveal a prominent role for the microbiota in shaping the transcriptional landscape of the hepatic endothelium., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)