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Layered double hydroxide (LDH) nanoparticles enable foliar delivery of genetic material, herbicides, and nutrients to promote plant growth and yield. Understanding the foliar uptake route of nanoparticles is needed to maximize their effectiveness and avoid unwanted negative effects. In this study, we investigated how delivering layered double hydroxide ( d = 37 ± 1.5 nm) through the adaxial (upper) or abaxial (lower) side of leaves affects particle uptake, nutrient delivery, and photosynthesis in tomato plants. LDH applied on the adaxial side was embedded in the cuticle and accumulated at the anticlinal pegs between epidermal cells. On the abaxial side, LDH particles penetrated the cuticle less, but the presence of the stomata enables penetration to deeper leaf layers. Accordingly, the average penetration levels of LDH relative to the cuticle were 2.47 ± 0.07, 1.25 ± 0.13, and 0.75 ± 0.1 μm for adaxial, abaxial with stomata, and abaxial without stomata leaf segments, respectively. In addition, the colocalization of LDH with the cuticle was ∼2.3 times lower for the adaxial application, indicating the ability to penetrate the cuticle. Despite the low adaxial stomata density, LDH-mediated delivery of magnesium (Mg) from leaves to roots was 46% higher for the adaxial than abaxial application. In addition, adaxial application leads to ∼24% higher leaf CO 2 assimilation rate and higher biomass accumulation. The lower efficiency from the abaxial side was, at least partially, a result of interference with the stomata functionality which reduced stomatal conductance and evapotranspiration by 28% and 25%, respectively, limiting plant photosynthesis. This study elucidates how foliar delivery pathways through different sides of the leaves affect their ability to deliver active agents into plants and consequently affect the plants' physiological response. That knowledge enables a more efficient use of nanocarriers for agricultural applications., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Context: Obesity is a risk factor for coronavirus disease 2019 (COVID-19)-related outcomes; however, the mechanism remains unclear., Objective: The objective of this analysis was to determine whether inflammation mediates the association between obesity and COVID-19 outcomes., Methods: The International Study of Inflammation in COVID-19 (ISIC): A Prospective Multi-Center Observational Study Examining the Role of Biomarkers of Inflammation in Predicting Covid-19 Related Outcomes in Hospitalized Patients, was conducted at 10 hospitals in the United States and Europe. Participants were adults hospitalized specifically for COVID-19 between February 1, 2020, through October 19, 2022. Inflammatory biomarkers, including soluble urokinase plasminogen activator receptor (suPAR), were measured at admission. Associations were examined between body mass index (BMI, kg/m2) and a composite of death, need for mechanical ventilation, and renal replacement therapy, stratified by pre- and post-Omicron variants. The contribution of inflammation to the relationship between obesity and outcomes was assessed., Results: Among 4644 participants (mean age 59.3, 45.6% male, 21.8% BMI ≥ 35), those with BMI > 40 (n = 485) had 55% higher odds of the composite outcome (95% CI, 1.21-1.98) compared with nonobese individuals (BMI < 30, n = 2358) in multivariable analysis. In multiple mediation analysis, only suPAR remained a significant mediator between BMI and composite outcome. Associations were amplified for participants younger than 65 years and with pre-Omicron variants., Conclusion: Obesity is associated with worse outcomes in COVID-19, notably in younger participants and in the pre-Omicron era. Inflammation, as measured by suPAR, is a significant mediator of the association between obesity and COVID-19 outcomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Background: Platelets prevent bleeding in a variety of inflammatory settings, the adhesion receptors and activation pathways involved being highly context-dependent and functionally redundant. In some situations, platelets recruited to inflammatory sites act independently of aggregation. The mechanisms underlying stable platelet adhesion in inflamed microvessels remain incompletely understood, in particular, whether and if so, how β1 and β3 integrins are involved., Methods: The impact of isolated or combined platelet deficiency in β1 and β3 integrins on inflammation-associated hemostasis was investigated in 3 models of acute inflammation: immune complex-based cutaneous reverse passive Arthus reaction, intranasal lipopolysaccharide-induced lung inflammation, and cerebral ischemia-reperfusion following transient (2-hour) occlusion of the middle cerebral artery., Results: Mice with platelet-directed inactivation of Itgb1 (PF4Cre-β1 -/- ) displayed no bleeding in any of the inflammation models, while mice defective in platelet Itgb3 (PF4Cre-β3 -/- ) exhibited bleeding in all 3 models. Remarkably, the bleeding phenotype of PF4Cre-β3 -/- mice was exacerbated in the reverse passive Arthus model by the concomitant deletion of β1 integrins, PF4Cre-β1 -/- /β3 -/- animals presenting increased bleeding. Intravital microscopy in reverse passive Arthus experiments highlighted a major defect in the adhesion of PF4Cre-β1 -/- /β3 -/- platelets to inflamed microvessels. Unlike PF4Cre-β1 -/- and PF4Cre-β3 -/- mice, PF4Cre-β1 -/- /β3 -/- animals developed early hemorrhagic transformation 6 hours after transient middle cerebral artery occlusion. PF4Cre-β1 -/- /β3 -/- mice displayed no more bleeding in lipopolysaccharide-induced lung inflammation than PF4Cre-β3 -/- animals., Conclusions: Altogether, these results show that the requirement for and degree of functional redundancy between platelet β1 and β3 integrins in inflammation-associated hemostasis vary with the inflammatory situation., Competing Interests: None.

Lupus Nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE) that affects kidney function. Here, we investigated the role of CD11b, a protein encoded by the ITGAM gene, in the development of LN and its functional activation as a therapeutic strategy. Genetic coding variants of ITGAM significantly increase the risk for SLE and LN by producing a less active CD11b and leading to elevated levels of type I interferon (IFN I). However, a molecular mechanism for how these variants increase LN risk has been unclear. Here, we determined that these variants also significantly associate with elevations in soluble urokinase plasminogen activator receptor (suPAR), a known biomarker linked to kidney disease, suggesting a novel molecular connection. Pharmacologic activation of CD11b with a novel, clinical-stage agonist ONT01 significantly suppressed suPAR production in myeloid cells and reduced systemic inflammation and kidney damage in multiple experimental models of LN. Importantly, delaying treatment with ONT01 until after disease onset also significantly reduced serum suPAR and inflammatory cytokines, and decreased immune complex deposition in the glomerulus, glomerulonephritis and albuminuria, suggesting that CD11b activation is therapeutic for LN. Genetic activation of CD11b via a gain-of-function CD11b mutation also showed complete protection from LN, whereas genetic deletion of CD11b worsened the disease in mice, providing further evidence of the role of CD11b activation in regulating LN. Finally, transfer of human LN PBMCs generated human LN like disease in mice that was significantly reduced by ONT01. Together, these data provide strong evidence that ONT01 mediated CD11b activation can therapeutically modulate TLR7-driven inflammation and protect against LN. These findings support clinical development of CD11b agonists as novel therapeutics for treating lupus nephritis in human patients.

Multiple myeloma (MM) is a plasma cell disease with a preferential bone marrow (BM) tropism. Enforced expression of tissue-specific chemokine receptors has been shown to successfully guide adoptively-transferred CAR NK cells towards the malignant milieu in solid cancers, but also to BM-resident AML and MM. For redirection towards BM-associated chemokine CXCL12, we armored BCMA CAR-NK-92 as well as primary NK cells with ectopic expression of either wildtype CXCR4 or a gain-of-function mutant CXCR4 R334X . Our data showed that BCMA CAR-NK-92 and -primary NK cells equipped with CXCR4 gained an improved ability to migrate towards CXCL12 in vitro . Beyond its classical role coordinating chemotaxis, CXCR4 has been shown to participate in T cell co-stimulation, which prompted us to examine the functionality of CXCR4-cotransduced BCMA-CAR NK cells. Ectopic CXCR4 expression enhanced the cytotoxic capacity of BCMA CAR-NK cells, as evidenced by the ability to eliminate BCMA-expressing target cell lines and primary MM cells in vitro and through accelerated cytolytic granule release. We show that CXCR4 co-modification prolonged BCMA CAR surface deposition, augmented ZAP-70 recruitment following CAR-engagement, and accelerated distal signal transduction kinetics. BCMA CAR sensitivity towards antigen was enhanced by virtue of an enhanced ZAP-70 recruitment to the immunological synapse, revealing an increased propensity of CARs to become triggered upon CXCR4 overexpression. Unexpectedly, co-stimulation via CXCR4 occurred in the absence of CXCL12 ligand-stimulation. Collectively, our findings imply that co-modification of CAR-NK cells with tissue-relevant chemokine receptors affect adoptive NK cell therapy beyond improved trafficking and retention within tumor sites., Competing Interests: AR and UH filed a patent application for the BCMA CAR used in this manuscript PCT/WO2017211900A1. AR and UH receive research funds from Fate Therapeutics San Diego, CA. JR, KM, JG, and BV are employees of Fate Therapeutics. 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., (Copyright © 2024 Moles, Erdlei, Menzel, Massaro, Fiori, Bunse, Schrimpf, Gerlach, Gudipati, Reiser, Mathavan, Goodrich, Huppa, Krönke, Valamehr, Höpken and Rehm.)

Objectives: To evaluate the impact of intubation timing, guided by severity criteria, on mortality in critically ill COVID-19 patients, amidst existing uncertainties regarding optimal intubation practices., Design: Prospective, multicenter, observational study conducted from February 1, 2020, to November 1, 2022., Setting: Ten academic institutions in the United States and Europe., Patients: Adults (≥ 18 yr old) confirmed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and hospitalized specifically for COVID-19, requiring intubation postadmission. Exclusion criteria included patients hospitalized for non-COVID-19 reasons despite a positive SARS-CoV-2 test., Interventions: Early invasive mechanical ventilation (EIMV) was defined as intubation in patients with less severe organ dysfunction (Sequential Organ Failure Assessment [SOFA] < 7 or Pa o2 /F io2 ratio > 250), whereas late invasive mechanical ventilation (LIMV) was defined as intubation in patients with SOFA greater than or equal to 7 and Pa o2 /F io2 ratio less than or equal to 250., Measurements and Main Results: The primary outcome was mortality within 30 days of hospital admission. Among 4464 patients, 854 (19.1%) required mechanical ventilation (mean age 60 yr, 61.7% male, 19.3% Black). Of those, 621 (72.7%) were categorized in the EIMV group and 233 (27.3%) in the LIMV group. Death within 30 days after admission occurred in 278 patients (42.2%) in the EIMV and 88 patients (46.6%) in the LIMV group ( p = 0.28). An inverse probability-of-treatment weighting analysis revealed a statistically significant association with mortality, with patients in the EIMV group being 32% less likely to die either within 30 days of admission (adjusted hazard ratio [HR] 0.68; 95% CI, 0.52-0.90; p = 0.008) or within 30 days after intubation irrespective of its timing from admission (adjusted HR 0.70; 95% CI, 0.51-0.90; p = 0.006)., Conclusions: In severe COVID-19 cases, an early intubation strategy, guided by specific severity criteria, is associated with a reduced risk of death. These findings underscore the importance of timely intervention based on objective severity assessments., Competing Interests: Dr. Hayek is funded by the National Heart, Lung, and Blood Institute 1R01HL153384-01, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 1R01DK12801201A1, U01-DK119083-03S1, and the Frankel Cardiovascular Center COVID-19: Impact Research Ignitor (U-M G024231) award. Dr. Pop-Busui is supported by the National Institutes of Health (NIH)/NIDDK-1-R01-DK-107956-01, NIH U01 DK119083, the Juvenile Diabetes Research Foundation 5-COE-2019-861-S-B, and by a Pilot and Feasibility Grant from the Michigan Diabetes Research Center (NIH Grant P30-DK020572). Dr. Huang disclosed work for hire. Dr. Giamarellos-Bourboulis’ institution received funding from Abbott Products Operations AG, BioMerieux, and MSD; they received funding from Abbott CH, BioMérieux, Brahms GmbH, GSK, InflaRx GmbH, Sobi, XBiotech, AbbVie, Johnson & Johnson, MSD, Novartis, UCB, and from the Horizon2020 Marie-Curie Project European Sepsis Academy, the Horizon 2020 European Grants ImmunoSep and RISKinCOVID, and from the Horizon Europe project EPIC-CROWN-2. Drs. Reiser and Banerjee received support for article research from the NIH. Dr. Reiser disclosed they are a co-founder and co-chair of the Scientific Advisory Board of Walden Biosciences; they received support for article research from Rush University Medical Center. Dr. Banerjee’s institution received funding from the NIDDK; they received funding from Novo Nordisk, Roche, and Lexicon Pharmaceuticals. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.)

Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering., (© 2024. The Author(s).)

Background: Understanding the dynamics of conduction velocity (CV) and voltage amplitude (VA) is crucial in cardiac electrophysiology, particularly for substrate-based catheter ablations targeting slow conduction zones and low voltage areas. This study utilizes ultra-high-density mapping to investigate the impact of heart rate and pacing location on changes in the wavefront direction, CV, and VA of healthy pig hearts., Methods: We conducted in vivo electrophysiological studies on four healthy juvenile pigs, involving various pacing locations and heart rates. High-resolution electroanatomic mapping was performed during intrinsic normal sinus rhythm (NSR) and electrical pacing. The study encompassed detailed analyses at three levels: entire heart cavities, subregions, and localized 5-mm-diameter circular areas. Linear mixed-effects models were used to analyze the influence of heart rate and pacing location on CV and VA in different regions., Results: An increase in heart rate correlated with an increase in conduction velocity and a decrease in voltage amplitude. Pacing influenced conduction velocity and voltage amplitude. Pacing also influenced conduction velocity and voltage amplitude, with varying effects observed based on the pacing location within different heart cavities. Pacing from the right atrium (RA) decreased CV in all heart cavities. The overall CV and VA changes in the whole heart cavities were not uniformly reflected in all subregions and subregional CV and VA changes were not always reflected in the overall analysis. Overall, there was a notable variability in absolute CV and VA changes attributed to pacing., Conclusions: Heart rate and pacing location influence CV and VA within healthy juvenile pig hearts. Subregion analysis suggests that specific regions of the heart cavities are more susceptible to pacing. High-resolution mapping aids in detecting regional changes, emphasizing the substantial physiological variations in CV and VA.

Diabetic chronic ulcers are plagued with persistent nonresolving inflammation. However, diabetic wound environment early after injury suffers from inadequate inflammatory responses due to reductions in proinflammatory cytokines levels. Diabetic neutrophils have known impairments in bactericidal functions. We hypothesized that reduced bacterial killing by diabetic neutrophils, due to their bactericidal functional impairments, results in reduced bioactive bacterial products, known as pathogen-associated molecular patterns, which in turn contribute to reduced signaling through toll-like receptors, leading to inadequate production of proinflammatory cytokines in infected diabetic wound early after injury. We tested our hypothesis in db/db type 2 obese diabetic mouse wound infection model with Pseudomonas aeruginosa. Our data indicate that despite substantially higher levels of infection, toll-like receptor 4-mediated signaling is reduced in diabetic wounds early after injury owing to reduced bioactive levels of lipopolysaccharide. We further demonstrate that topical treatment with lipopolysaccharide enhances toll-like receptor 4 signaling, increases proinflammatory cytokine production, restores leukocyte trafficking, reduces infection burden, and stimulates healing in diabetic wounds. We posit that lipopolysaccharide may be a viable therapeutic option for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process, which is intended to reset chronic ulcers into acute fresh wounds., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Inflammatory demyelinating diseases of the central nervous system, such as multiple sclerosis, are significant sources of morbidity in young adults despite therapeutic advances. Current murine models of remyelination have limited applicability due to the low white matter content of their brains, which restricts the spatial resolution of diagnostic imaging. Large animal models might be more suitable but pose significant technological, ethical and logistical challenges., Methods: We induced targeted cerebral demyelinating lesions by serially repeated injections of lysophosphatidylcholine in the minipig brain. Lesions were amenable to follow-up using the same clinical imaging modalities (3T magnetic resonance imaging, 11 C-PIB positron emission tomography) and standard histopathology protocols as for human diagnostics (myelin, glia and neuronal cell markers), as well as electron microscopy (EM), to compare against biopsy data from two patients., Findings: We demonstrate controlled, clinically unapparent, reversible and multimodally trackable brain white matter demyelination in a large animal model. De-/remyelination dynamics were slower than reported for rodent models and paralleled by a degree of secondary axonal pathology. Regression modelling of ultrastructural parameters (g-ratio, axon thickness) predicted EM features of cerebral de- and remyelination in human data., Interpretation: We validated our minipig model of demyelinating brain diseases by employing human diagnostic tools and comparing it with biopsy data from patients with cerebral demyelination., Funding: This work was supported by the DFG under Germany's Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy, ID 390857198) and TRR 274/1 2020, 408885537 (projects B03 and Z01)., Competing Interests: Declaration of interests CS, SH, BL, EL and TL are part of Ergosurg GmbH, which developed and manufactured the navigation system, the trackable instruments and the robotic system. VMB has received consulting fees from Brainlab. IY has received grants from the German Federal Ministry of Education and Research (BMBF) and the German Research Foundation (DFG), consulting fees from ABX-CRO, Blue Earth Diagnostics and Pentixapharm, honoraria from Piramal, support for attending meeting from the Society of Nuclear Medicine and Molecular Imaging, the European Association of Nuclear Medicine, the Slovenian Neuroscience Association (SiNAPSA) and the International Brain Research Organization, and is a member of the Neuroimaging Committee, European Association of Nuclear Medicine, the Board of Directors, Brain Imaging Council, Society of Nuclear Medicine and Molecular Imaging as well as the Molecular Connectivity Working Group. JK has received consulting fees from Novartis, possesses stock options at Bonescreen GmbH and was supported by the European Research Council, the DFG and the BMBF. TM has received speaker fees from Novartis and Roche as well as travel support from Novartis. BH has received consulting fees from GLG Consulting, Sandoz and Polpharma, possesses issued patents for detection of antibodies against KIR4.1 in a subpopulation of patients with multiple sclerosis, as well as genetic determinants of neutralizing antibodies to interferon, and has participated on Data Safety Monitoring and Advisory Boards for Novartis, Sandoz, Polpharma, Allergycare, TG Therapeutics and Biocon., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Soluble urokinase plasminogen activator receptor is an inflammatory biomarker that may prognosticate cardiovascular outcomes. We sought to determine the associations between soluble urokinase plasminogen activator receptor and established markers of cardiotoxicity in breast cancer patients receiving doxorubicin., Methods: We conducted a prospective cohort study of women with newly diagnosed breast cancer receiving standard-dose doxorubicin (240 mg/m 2 ) at Rush University Medical Center and Rush Oak Park Hospital (Chicago, IL) between January 2017 and May 2019. Left ventricular ejection fraction, global longitudinal strain, and cardiac biomarkers (N-terminal prohormone B-type natriuretic peptide, troponin-I, and high-sensitivity C-reactive protein) were measured at baseline and at intervals up to 12-month follow-up after end of treatment. The associations between soluble urokinase plasminogen activator receptor and these endpoints were evaluated using multivariable mixed effects linear regression., Results: Our study included 37 women (mean age 47.0 ± 9.3 years, 60% white) with a median baseline soluble urokinase plasminogen activator receptor level of 2.83 ng/dL. No participant developed cardiomyopathy based on serial echocardiography by one-year follow-up. The median percent change in left ventricular strain was -4.3% at 6-month follow-up and absolute changes in cardiac biomarkers were clinically insignificant. There were no significant associations between soluble urokinase plasminogen activator receptor and these markers of cardiotoxicity (all p > 0.05)., Conclusions: In this breast cancer cohort, doxorubicin treatment was associated with a very low risk for cardiotoxicity. Across this narrow range of clinical endpoints, soluble urokinase plasminogen activator receptor was not associated with markers of subclinical cardiotoxicity. Further studies are needed to clarify the prognostic utility of soluble urokinase plasminogen activator receptor in doxorubicin-associated cardiomyopathy and should include a larger cohort of leukemia and lymphoma patients who receive higher doses of doxorubicin., (© 2024. The Author(s).)

Severe coronavirus disease 2019 (COVID-19) is a hyperinflammatory syndrome. The biomarkers of inflammation best suited to triage patients with COVID-19 are unknown. We conducted a prospective multicenter observational study of adult patients hospitalized specifically for COVID-19 from February 1, 2020 to October 19, 2022. Biomarkers measured included soluble urokinase plasminogen activator receptor (suPAR), C-reactive protein, interleukin-6, procalcitonin, ferritin, and D-dimer. In-hospital outcomes examined include death and the need for mechanical ventilation. Patients admitted in the United States (US, n = 1962) were used to compute area under the curves (AUCs) and identify biomarker cutoffs. The combined European cohorts (n = 1137) were used to validate the biomarker cutoffs. In the US cohort, 356 patients met the composite outcome of death (n = 197) or need for mechanical ventilation (n = 290). SuPAR was the most important predictor of the composite outcome and had the highest AUC (0.712) followed by CRP (0.642), ferritin (0.619), IL-6 (0.614), D-dimer (0.606), and lastly procalcitonin (0.596). Inclusion of other biomarkers did not improve discrimination. A suPAR cutoff of 4.0 ng/mL demonstrated a sensitivity of 95.4% (95% CI: 92.4%-98.0%) and negative predictive value (NPV) of 92.5% (95% CI: 87.5%-96.9%) for the composite outcome. Patients with suPAR < 4.0 ng/mL comprised 10.6% of the cohort and had a 0.8% probability of the composite outcome. Applying this cutoff to the validation cohort yielded a sensitivity of 93.8% (90.4%-96.7%) and NPV of 95.5% (93.1%-97.8%) for the composite outcome. Among commonly measured biomarkers, suPAR offered stronger discriminatory ability and may be useful in triaging low-risk patients with COVID-19., (© 2024 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Objective: Due to severely limited donor heart availability, durable mechanical circulatory support remains the only treatment option for many patients with end-stage heart failure. However, treatment complexity persists due to its univentricular support modality and continuous contact with blood. We investigated the function and safety of reBEAT (AdjuCor GmbH), a novel, minimal invasive mechanical circulatory support device that completely avoids blood contact and provides pulsatile, biventricular support., Methods: For each animal tested, an accurately sized cardiac implant was manufactured from computed tomography scan analyses. The implant consists of a cardiac sleeve with three inflatable cushions, 6 epicardial electrodes and driveline connecting to an electro-pneumatic, extracorporeal portable driver. Continuous epicardial electrocardiogram signal analysis allows for systolic and diastolic synchronization of biventricular mechanical support. In 7 pigs (weight, 50-80 kg), data were analyzed acutely (under beta-blockade, n = 5) and in a 30-day long-term survival model (n = 2). Acquisition of intracardiac pressures and aortic and pulmonary flow data were used to determine left ventricle and right ventricle stroke work and stroke volume, respectively., Results: Each implant was successfully positioned around the ventricles. Automatic algorithm electrocardiogram signal annotations resulted in precise, real-time mechanical support synchronization with each cardiac cycle. Consequently, progressive improvements in cardiac hemodynamic parameters in acute animals were achieved. Long-term survival demonstrated safe device integration, and clear and stable electrocardiogram signal detection over time., Conclusions: The present study demonstrates biventricular cardiac support with reBEAT. Various demonstrated features are essential for realistic translation into the clinical setting, including safe implantation, anatomical fit, safe device-tissue integration, and real-time electrocardiogram synchronized mechanical support, result in effective device function and long-term safety., (Copyright © 2022 The American Association for Thoracic Surgery. All rights reserved.)

Soluble urokinase plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. In addition to suPAR, proteolysis of membrane-bound uPAR results in circulating D1 and D2D3 proteins. We showed that when exposed to a high-fat diet, transgenic mice expressing D2D3 protein developed progressive kidney disease marked by microalbuminuria, elevated serum creatinine, and glomerular hypertrophy. D2D3 transgenic mice also exhibited insulin-dependent diabetes mellitus evidenced by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic β cell mass, and high fasting blood glucose. Injection of anti-uPAR antibody restored β cell mass and function in D2D3 transgenic mice. At the cellular level, the D2D3 protein impaired β cell proliferation and inhibited the bioenergetics of β cells, leading to dysregulated cytoskeletal dynamics and subsequent impairment in the maturation and trafficking of insulin granules. D2D3 protein was predominantly detected in the sera of patients with nephropathy and insulin-dependent diabetes mellitus. These sera inhibited glucose-stimulated insulin release from human islets in a D2D3-dependent manner. Our study showed that D2D3 injures the kidney and pancreas and suggests that targeting this protein could provide a therapy for kidney diseases and insulin-dependent diabetes mellitus.

Introduction: Soluble urokinase plasminogen activation receptor (suPAR) is an immune-derived pathogenic factor for kidney and atherosclerotic disease. Whether the association between suPAR and cardiovascular (CV) outcomes is dependent on the severity of underlying kidney disease is unclear., Methods: We measured serum suPAR levels in 4994 participants (mean age 60 years; 60% men; 36% with diabetes mellitus; mean estimated glomerular filtration rate (eGFR) 49 ml/min per 1.73 m 2 , SD 18) of the German Chronic Kidney Disease (GCKD) cohort and examined its association with all-cause death, CV death, and major CV events (MACE) across the range of eGFR and urine albumin-to-creatinine ratio (UACR)., Results: The median suPAR level was 1771 pg/ml (interquartile range [IQR] 1447-2254 pg/ml). SuPAR levels were positively and independently correlated with age, eGFR, UACR, and parathyroid hormone levels. There were 573 deaths, including 190 CV deaths and 683 MACE events at a follow-up time of 6.5 years. In multivariable analyses, suPAR levels (log 2 ) were associated with all-cause death (hazard ratio [HR] 1.36, 95% confidence interval [CI] 1.21-1.53), CV death (HR 1.27, 95% CI 1.03-1.57), and MACE (HR 1.13, 95% CI 1.00-1.28), and were not found to differ according to diabetes mellitus status, baseline eGFR, UACR, or parathyroid hormone levels. In mediation analysis, suPAR's direct effect on all-cause death, CV death, and MACE accounted for 77%, 67%, and 60% of the total effect, respectively; whereas the effect mediated through eGFR accounted for 23%, 34%, and 40%, respectively., Conclusion: In a large cohort of individuals with chronic kidney disease (CKD), suPAR levels were associated with mortality and CV outcomes independently of indices of kidney function, consistent with its independent role in the pathogenesis of atherosclerosis., (© 2023 International Society of Nephrology. Published by Elsevier Inc.)

Many potentially noxious interventions are performed on chicken embryos in research and in the poultry industry. It is therefore essential and in the interest of animal welfare to be able to precisely define the point at which a chicken embryo is capable of nociception in ovo. The present part III of a comprehensive study examined the movements of developing chicken embryos with the aim of identifying behavioral responses to a noxious stimulus. For this purpose, a noxious mechanical stimulus and a control stimulus were applied in a randomized order. The recorded movements of the embryos were evaluated using the markerless pose estimation software DeepLabCut and manual observations. After the application of the mechanical stimulus, a significant increase in beak movement was identified in 15- to 18-day-old embryos. In younger embryos, no behavioral changes related to the noxious stimulus were observed. The presented results indicate that noxious mechanical stimuli at the beak base evoke a nocifensive reaction in chicken embryos starting at embryonic day 15.

Chicken culling has been forbidden in Germany since 2022; male/female selection and male elimination must be brought to an embryonic status prior to the onset of nociception. The present study evaluated the ontogenetic point at which noxious stimuli could potentially be perceived/processed in the brain in ovo . EEG recordings from randomized hyperpallial brain sites were recorded in ovo and noxious stimuli were applied. Temporal and spectral analyses of the EEG were performed. The onset of physiological neuronal signals could be determined at developmental day 13. ERP/ERSP/ITC analysis did not reveal phase-locked nociceptive responses. Although no central nociceptive responses were documented, adequate EEG responses to noxious stimuli from other brain areas cannot be excluded. The extreme stress impact on the embryo during the recording may overwrite the perception of noniceptive stimuli. The results suggest developmental day 13 as the earliest embryonal stage being able to receive and process nociceptive stimuli.

Background: Ultra-high-density mapping systems allow more precise measurement of the heart chambers at corresponding conduction velocities (CVs) and voltage amplitudes (VAs). Our aim for this study was to define and compare a basic value set for unipolar CV and VA in all four heart chambers and their separate walls in healthy, juvenile porcine hearts using ultra-high-density mapping., Methods: We used the Rhythmia Mapping System to create electroanatomical maps of four pig hearts in sinus rhythm. CVs and VAs were calculated for chambers and wall segments with overlapping circular areas (radius of 5 mm)., Results: We analysed 21 maps with a resolution of 1.4 points/mm 2 . CVs were highest in the left atrium (LA), followed by the left ventricle (LV), right ventricle (RV), and right atrium (RA). As for VA, LV was highest, followed by RV, LA, and RA. The left chambers had a higher overall CV and VA than the right. Within the chambers, CV varied more in the right than in the left chambers, and VA varied in the ventricles but not in the atria. There was a slightly positive correlation between CVs and VAs at velocity values of <1.5 m/s., Conclusions: In healthy porcine hearts, the left chambers showed higher VAs and CVs than the right. CV differs mainly within the right chambers and VA differs only within the ventricles. A slightly positive linear correlation was found between slow CVs and low VAs.

Although it is assumed that chicken embryos acquire the capacity for nociception while developing in the egg, an exact time point has not yet been specified. The present research was an exploratory study aiming to determine when the capacity of nociception emerges during embryonic development in chickens. Changes in blood pressure and heart rate (HR) in response to a noxious mechanical stimulus at the base of the beak versus a light touch on the beak were examined in chicken embryos between embryonic days (EDs) 7 and 18. Mean arterial pressure (MAP) was the most sensitive parameter for assessing cardiovascular responses. Significant changes in MAP in response to a noxious stimulus were detected in embryos at ED16 to ED18, whereas significant changes in HR were observed at ED17 and ED18. Infiltration anesthesia with the local anesthetic lidocaine significantly reduced the response of MAP on ED18, so the measured cardiovascular changes may be interpreted as nociceptive responses.

Elevation in soluble urokinase receptor (suPAR) and proteinuria are common signs in patients with moderate to severe coronavirus disease 2019 (COVID-19). Here we characterize a new type of proteinuria originating as part of a viral response. Inoculation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes increased suPAR levels and glomerulopathy in African green monkeys. Using an engineered mouse model with high suPAR expression, inhaled variants of SARS-CoV-2 spike S1 protein elicite proteinuria that could be blocked by either suPAR antibody or SARS-CoV-2 vaccination. In a cohort of 1991 COVID-19 patients, suPAR levels exhibit a stepwise association with proteinuria in non-Omicron, but not in Omicron infections, supporting our findings of biophysical and functional differences between variants of SARS-CoV-2 spike S1 protein and their binding to podocyte integrins. These insights are not limited to SARS-CoV-2 and define viral response proteinuria (VRP) as an innate immune mechanism and co-activation of podocyte integrins., (© 2023. The Author(s).)

Objective: This study assessed the use of a topical anesthetic as a feasible approach to reduce pain during piglet castration using a minimal anesthesia protocol., Animals: 18 male piglets, aged 3-6 days, were included in this study., Methods: A minimal anesthetic state was induced with isoflurane administered by facemask, with anesthetic depth individually adjusted based on responses to interdigital pinch. To desensitize the scrotal skin, a vapocoolant was applied 3 times. Scrotal incisions were made subsequently and Tri-Solfen (TS) or Placebo (P) was administered in both incisional gaps. After 30 seconds, the spermatic cords were severed followed by a further application of TS/P to both incision edges. Nociception-related variables, such as mean arterial blood pressure (MAP), heart rate (HR), and nocifensive movements, were assessed., Results: Significant differences in MAP changes were assessed between the TS (14 ± 4 mmHg) and the P group (36 ± 8 mmHg) for cutting the spermatic cords. Furthermore, significantly fewer nocifensive movement score points appeared in the TS than in the P group (0; IQR = 0 vs 5; IQR = 6)., Clinical Relevance: In this anesthesia model, the application of TS after skin incision significantly reduced MAP responses and nocifensive movements with spermatic cord transection compared with the application of P. However, the waiting period between TS-application and spermatic cord transection might limit the benefit of the method in conscious piglets, as pain during castration is reduced but additional stress is caused by the prolonged handling. Furthermore, using a vapocoolant did not provide anesthesia for skin incisions.

Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as TTLL12 , which is an inhibitor of antiviral innate immunity may be introduced and multiplexed to yield cell lines with improved LV vector productivity., 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 Iaffaldano, Marino and Reiser.)

Background: The administration of modified immune cells (MIC) before kidney transplantation led to specific immunosuppression against the allogeneic donor and a significant increase in regulatory B lymphocytes. We wondered how this approach affected the continued clinical course of these patients., Methods: Ten patients from a phase I clinical trial who had received MIC infusions prior to kidney transplantation were retrospectively compared to 15 matched standard-risk recipients. Follow-up was until year five after surgery., Results: The 10 MIC patients had an excellent clinical course with stable kidney graft function, no donor-specific human leukocyte antigen antibodies (DSA) or acute rejections, and no opportunistic infections. In comparison, a retrospectively matched control group receiving standard immunosuppressive therapy had a higher frequency of DSA (log rank P = 0.046) and more opportunistic infections (log rank P = 0.033). Importantly, MIC patients, and in particular the four patients who had received the highest cell number 7 days before surgery and received low immunosuppression during follow-up, continued to show a lack of anti-donor T lymphocyte reactivity in vitro and high CD19 + CD24 hi CD38 hi transitional and CD19 + CD24 hi CD27 + memory B lymphocytes until year five after surgery., Conclusions: MIC infusions together with reduced conventional immunosuppression were associated with good graft function during five years of follow-up, no de novo DSA development and no opportunistic infections. In the future, MIC infusions might contribute to graft protection while reducing the side effects of immunosuppressive therapy. However, this approach needs further validation in direct comparison with prospective controls., Trial Registration: https://clinicaltrials.gov/, identifier NCT02560220 (for the TOL-1 Study). EudraCT Number: 2014-002086-30., Competing Interests: MSa, CMo, CK, GO, MZ, MSm, PT, and ASm together with the University of Heidelberg, are co-founders of TolerogenixX GmbH, Heidelberg, Germany, a biotechnology company that holds licenses for MIC treatment. CK, GO, and PT hold a patent for MIC treatment. MSa, CMo, CK, GO, CSü, MZ, MSm, PT, AS, and VD together with the University of Heidelberg and TolerogenixX GmbH filed a patent application for MIC treatment. 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., (Copyright © 2023 Schaier, Morath, Wang, Kleist, Opelz, Tran, Scherer, Pham, Ekpoom, Süsal, Ponath, Kälble, Speer, Benning, Nusshag, Mahler, Pego da Silva, Sommerer, Hückelhoven-Krauss, Czock, Mehrabi, Schwab, Waldherr, Schnitzler, Merle, Schwenger, Krautter, Kemmner, Fischereder, Stangl, Hauser, Kälsch, Krämer, Böhmig, Müller-Tidow, Reiser, Zeier, Schmitt, Terness, Schmitt and Daniel.)

Background: Immunotherapies are becoming front-line treatments for many advanced cancers, and combinations of two or more therapies are beginning to be investigated. Based on their individual antitumor capabilities, we sought to determine whether combination oncolytic virus (OV) and radiation therapy (RT) may improve cancer outcomes., Methods: To investigate the activity of this combination therapy, we used in vitro mouse and human cancer cell lines as well as a mouse model of skin cancer. After initial results, we further included immune checkpoint blockade, whose addition constituted a triple combination immunotherapy., Results: Our findings demonstrate that OV and RT reduce tumor growth via conversion of immunologically 'cold' tumors to 'hot', via a CD8+ T cell-dependent and IL-1α-dependent mechanism that is associated with increased PD-1/PD-L1 expression, and the triple combination of OV, RT, and PD-1 checkpoint inhibition impedes tumor growth and prolongs survival. Further, we describe the response of a PD-1-refractory patient with cutaneous squamous cell carcinoma who received the triple combination of OV, RT, and immune checkpoint inhibitor (ICI), and went on to experience unexpected, prolonged control and survival. He remains off-treatment and is without evidence of progression for >44 months since study entry., Conclusions: Effective systemic antitumor immune response is rarely elicited by a single therapy. In a skin cancer mouse model, we demonstrate improved outcomes with combination OV, RT, and ICI treatment, which is associated with mechanisms involving augmented CD8+ T cell infiltration and IL-1α expression. We report tumor reduction and prolonged survival of a patient with skin cancer treated with combination OV, RT, and ICI. Overall, our data provide strong rationale for combining OV, RT, and ICI for treatment of patients with ICI-refractory skin and potentially other cancers., Competing Interests: Competing interests: HLK is an employee of Ankyra Therapeutics and has served on advisory boards for Castle Biosciences, Marengo Therapeutics, and Virogin. AWS reports receiving grants/research support (to the Institution) from Biohaven Pharmaceuticals, Replimune, Morphogenesis, Shattuck Laboratories, advisory board fees from InStil Bio and Signatera, and royalties from UpToDate. AZ holds equity in Primevax Immuno-Oncology., (© 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.)

Background: Focal segmental glomerulosclerosis (FSGS) is frequently associated with heavy proteinuria and progressive renal failure requiring dialysis or kidney transplantation. However, primary FSGS also has a ~40% risk of recurrence of disease in the transplanted kidney (rFSGS). Multiple circulating factors have been identified to contribute to the pathogenesis of primary and rFSGS including soluble urokinase-type plasminogen activator receptor (suPAR) and patient-derived CD40 autoantibody (CD40autoAb). However, the downstream effector pathways specific to individual factors require further study. The tumor necrosis factor, TNF pathway activation by one or more circulating factors present in the sera of patients with FSGS has been supported by multiple studies., Methods: A human in vitro model was used to study podocyte injury measured as the loss of actin stress fibers. Anti-CD40 autoantibody was isolated from FSGS patients (recurrent and non-recurrent) and control patients with ESRD due to non-FSGS related causes. Two novel human antibodies-anti-uPAR (2G10) and anti-CD40 antibody (Bristol Meyer Squibb, 986090) were tested for their ability to rescue podocyte injury. Podocytes treated with patient derived antibody were transcriptionally profiled using whole human genome microarray., Results: Here we show that podocyte injury caused by sera from FSGS patients is mediated by CD40 and suPAR and can be blocked by human anti-uPAR and anti-CD40 antibodies. Transcriptomic studies to compare the molecules and pathways activated in response to CD40 autoantibody from rFSGS patients (rFSGS/CD40autoAb) and suPAR, identified unique inflammatory pathways associated with FSGS injury., Conclusions: We identified several novel and previously described genes associated with FSGS progression. Targeted blockade of suPAR and CD40 pathways with novel human antibodies showed inhibition of podocyte injury in FSGS., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-3670/coif). MMS is a founder of technology for the assessment of kidney injury, owned by the regents, University of California. As the founder she has financial or non-financial interests in Nephrosant. JR reports that he receives consulting fees from Reata, Novateur Ventures, Walden Biosciences, Biomarin, Astellas, Massachusetts General Hospital, Genentech, Up to Date, Merck, Insceptionsci, GL, Visterra, Aclipse and MantraBio. JR also has several patents: US20110212083-Role of soluble uPAR in the Pathogenesis of Proteinuric Kidney Disease, S9867923-Reducing Solucble Rokinase Receptor in the Circulation, JP2016530510-Non-Glycoslyated suPAR Biomarkers and Uses thereof, US20160296592-Methods/Compositions for the Treatment of Proteinuric Diseases, US9144594-Dynamin Mediated Diseases and US8809386-Dynamin Ring Stabilizers. JR is on Scientific advisory board of Walden Biosciences and has stocks/stock options at Walden Biosciences and Aclipse. The other authors have no conflicts of interest to declare., (2023 Annals of Translational Medicine. All rights reserved.)

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

This work introduces a novel setup for computed tomography of heavy and bulky specimens at the SYRMEP beamline of the Italian synchrotron Elettra. All the key features of the setup are described and the first application to off-center computed tomography scanning of a human chest phantom (approximately 45 kg) as well as the first results for vertical helical acquisitions are discussed., (open access.)

Acute kidney injury (AKI) secondary to sepsis results in poor outcomes and conventional kidney function indicators lack diagnostic value. Soluble urokinase plasminogen activator receptor (suPAR) is an innate immune-derived molecule implicated in inflammatory organ damage. We characterized the diagnostic ability of longitudinal serum suPAR levels to discriminate severity and course of sepsis-induced AKI (SI-AKI) in 200 critically ill patients meeting Sepsis-3 criteria. The pathophysiologic relevance of varying suPAR levels in SI-AKI was explored in a polymicrobial sepsis model in WT, (s)uPAR-knockout, and transgenic suPAR-overexpressing mice. At all time points studied, suPAR provided a robust classification of SI-AKI disease severity, with improved prediction of renal replacement therapy (RRT) and mortality compared with established kidney biomarkers. Patients with suPAR levels of greater than 12.7 ng/mL were at highest risk for RRT or death, with an adjusted odds ratio of 7.48 (95% CI, 3.00-18.63). suPAR deficiency protected mice against SI-AKI. suPAR-overexpressing mice exhibited greater kidney damage and poorer survival through inflamed kidneys, accompanied by local upregulation of potent chemoattractants and pronounced kidney T cell infiltration. Hence, suPAR allows for an innate immune-derived and kidney function-independent staging of SI-AKI and offers improved longitudinal risk stratification. suPAR promotes T cell-based kidney inflammation, while suPAR deficiency improves SI-AKI.

Surgical castration of piglets is generally recognized as a painful procedure, but there is currently no gold standard for the assessment of pain behavior in piglets. However, pain assessment is essential for evaluating the effectiveness of local anesthetics. In this study, we investigated the efficacy of four local anesthetics in terms of pain relief during and after surgical castration in three sequential study parts. To do so, we filmed 178 piglets before the applied procedures, after injection of the local anesthetic, and up to 24 h after castration (five observation times in total) in an observation arena and compared their behavior before and after castration and between treatments and control groups. The results showed significant differences in the behavior of the piglets before and after castration and between the sham-castrated control group and the control group castrated without anesthesia. The different local anesthesia treatment groups showed diverging differences to the control groups. The most frequently shown pain-associated behaviors of the piglets were changes in tail position and hunched back posture. We observed a reduction but no complete elimination of the expressed pain-associated behaviors after local anesthesia. Several behavioral changes-such as changes in tail position, hunched back posture or tail wagging-persisted until the day after castration. Owing to the limited duration of the effects of the local anesthetics, local anesthesia did not influence long-term pain.

Objective: We demonstrate and discuss the use of mobile electroencephalogram (EEG) for neuroergonomics. Both technical state of the art as well as measures and cognitive concepts are systematically addressed., Background: Modern work is increasingly characterized by information processing. Therefore, the examination of mental states, mental load, or cognitive processing during work is becoming increasingly important for ergonomics., Results: Mobile EEG allows to measure mental states and processes under real live conditions. It can be used for various research questions in cognitive neuroergonomics. Besides measures in the frequency domain that have a long tradition in the investigation of mental fatigue, task load, and task engagement, new approaches-like blink-evoked potentials-render event-related analyses of the EEG possible also during unrestricted behavior., Conclusion: Mobile EEG has become a valuable tool for evaluating mental states and mental processes on a highly objective level during work. The main advantage of this technique is that working environments don't have to be changed while systematically measuring brain functions at work. Moreover, the workflow is unaffected by such neuroergonomic approaches.

Rationale, Aims and Objectives: Critics have charged that evidence-based medicine (EBM) overemphasises algorithmic rules over unstructured clinical experience and intuition, but the role of structured decision support systems in improving health outcomes remains uncertain. We aim to assess if delivery of anticoagulant prophylaxis in hospitalised patients with COVID-19 according to an algorithm based on evidence-based clinical practice guideline (CPG) improved clinical outcomes compared with administration of anticoagulant treatment given at individual practitioners' discretion., Methods: An observational design consisting of the analysis of all acutely ill, consecutive patients (n = 1783) with confirmed COVID-19 diagnosis admitted between 10 March 2020 to 11 January 2022 to an US academic center. American Society of Haematology CPG for anticoagulant prophylaxis in hospitalised patients with COVID-19 was converted into a clinical pathway and translated into fast-and-frugal decision (FFT) tree ('algorithm'). We compared delivery of anticoagulant prophylaxis in hospitalised patients with COVID-19 according to the FFT algorithm with administration of anticoagulant treatment given at individual practitioners' discretion., Results: In an adjusted analysis, using combination of Lasso (least absolute shrinkage and selection operator) and propensity score based weighting [augmented inverse-probability weighting] statistical techniques controlling for cluster data, the algorithm did not reduce death, venous thromboembolism, or major bleeding, but helped avoid longer hospital stay [number of patients needed to be treated (NNT) = 40 (95% CI: 23-143), indicating that for every 40 patients (23-143) managed on FFT algorithm, one avoided staying in hospital longer than 10 days] and averted admission to intensive-care unit (ICU) [NNT = 19 (95% CI: 13-40)]. All model's selected covariates were well balanced. The results remained robust to sensitivity analyses used to test the stability of the findings., Conclusions: When delivered using a structured FFT algorithm, CPG shortened the hospital stay and help avoided admission to ICU, but it did not affect other relevant outcomes., (© 2022 The Authors. Journal of Evaluation in Clinical Practice published by John Wiley & Sons Ltd.)

The impact of treatment on the outcome of chronic myelomonocytic leukemia (CMML) patients over a longer period of time and the potential role of predictive factors are not well defined. In a retrospective observational study, we analyzed 168 CMML patients regarding treatment options and survival during the past three decades. The proportion of patients treated with hydroxyurea (HU), intensive chemotherapy, and azacitidine (AZA) was 65/19/0% before 2000, 51/25/32% from 2000-2010, and 36/12/53% after 2010, respectively. Median overall survival (OS) increased from 10 months before 2000 to 23 months thereafter (p = 0.021). AZA-treated patients but not patients treated with other treatment options had improved survival as compared to CMML patients without AZA therapy (19 vs. 25 months, p = 0.041). When looking at subgroups, the following patient cohorts had a significant survival benefit in association with AZA therapy: patients with Hb > 10 g/dL, patients with monocytosis > 10 G/L, and patients with mutations in RASopathy genes., (© 2022. The Author(s).)

Background: Soluble urokinase-type plasminogen activator receptor (suPAR) is a marker of immune activation and pathogenic factor for kidney disease shown to predict cardiovascular outcomes including heart failure (HF) in various populations. We characterized suPAR levels in patients with HF and compared its ability to discriminate risk to that of B-type natriuretic peptide (BNP)., Methods and Results: We measured plasma suPAR and BNP levels in 3,437 patients undergoing coronary angiogram and followed for a median of 6.2 years. We performed survival analyses for the following outcomes: all-cause death, cardiovascular death, and hospitalization for HF. We then assessed suPAR's ability to discriminate risk for the aforementioned outcomes. We identified 1116 patients with HF (age 65±12, 67.2% male, 20.0% Black, 67% with reduced ejection fraction). The median suPAR level was higher in HF compared to those without HF (3370 [IQR 2610-4371] vs. 2880 [IQR 2270-3670] pg/mL, respectively, P<0.001). In patients with HF, suPAR levels (log-base 2) were associated with outcomes including all-cause death (adjusted hazard ratio aHR 2.30, 95%CI[1.90-2.77]), cardiovascular death (aHR 2.33 95%CI[1.81-2.99]) and HF hospitalization (aHR 1.96, 95%CI[1.06-1.25]) independently of clinical characteristics and BNP levels. The association persisted across subgroups and did not differ between patients with reduced or preserved ejection fraction, or those with ischemic or non-ischemic cardiomyopathy. Addition of suPAR to a model including BNP levels significantly improved the C-statistic for death (Δ0.027), cardiovascular death (Δ0.017) and hospitalization for HF (Δ0.017)., Conclusions: SuPAR levels are higher in HF compared to non-HF, are strongly predictive of outcomes, and combined with BNP, significantly improved risk prediction., Competing Interests: Conflict of Interest None of the authors have conflicts of interest to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: We recently demonstrated that donor-derived modified immune cells (MICs)-PBMCs that acquire immunosuppressive properties after a brief treatment-induced specific immunosuppression against the allogeneic donor when administered before kidney transplantation. We found up to a 68-fold increase in CD19 + CD24 hi CD38 hi transitional B lymphocytes compared with transplanted controls., Methods: Ten patients from a phase 1 clinical trial who had received MIC infusions before kidney transplantation were followed to post-transplant day 1080., Results: Patients treated with MICs had a favorable clinical course, showing no donor-specific human leukocyte antigen antibodies or acute rejections. The four patients who had received the highest dose of MICs 7 days before surgery and were on reduced immunosuppressive therapy showed an absence of in vitro lymphocyte reactivity against stimulatory donor blood cells, whereas reactivity against third party cells was preserved. In these patients, numbers of transitional B lymphocytes were 75-fold and seven-fold higher than in 12 long-term survivors on minimal immunosuppression and four operationally tolerant patients, respectively ( P <0.001 for both). In addition, we found significantly higher numbers of other regulatory B lymphocyte subsets and a gene expression signature suggestive of operational tolerance in three of four patients. In MIC-treated patients, in vitro lymphocyte reactivity against donor blood cells was restored after B lymphocyte depletion, suggesting a direct pathophysiologic role of regulatory B lymphocytes in donor-specific unresponsiveness., Conclusions: These results indicate that donor-specific immunosuppression after MIC infusion is long-lasting and associated with a striking increase in regulatory B lymphocytes. Donor-derived MICs appear to be an immunoregulatory cell population that when administered to recipients before transplantation, may exert a beneficial effect on kidney transplants., Clinical Trial Registry Name and Registration Number: MIC Cell Therapy for Individualized Immunosuppression in Living Donor Kidney Transplant Recipients (TOL-1), NCT02560220., (Copyright © 2022 by the American Society of Nephrology.)

People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR's pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the plasminogen activator, urokinase receptor (PLAUR) gene (rs4760), confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, proprotein convertase subtilisin/kexin-9 (Pcsk9) transfection in mice overexpressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared with those in WT mice, despite similar cholesterol levels. Prior to induction of atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared with WT aortas. Aortic and circulating suPARTg monocytes exhibited a proinflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.

Introduction: Donor-derived modified immune cells (MIC) induced long-term specific immunosuppression against the allogeneic donor in preclinical models of transplantation. In a phase I clinical trial (TOL-1 Study), MIC treatment resulted in a cellular phenotype that was directly and indirectly suppressive to the recipient's immune system allowing for reduction of conventional immunosuppressive therapy. Here, we describe a protocol for a randomised controlled, multicentre phase-IIb clinical trial of individualised immunosuppression with intravenously administered donor MIC compared with standard-of-care (SoC) in living donor kidney transplantation (TOL-2 Study)., Methods and Analysis: Sixty-three living donor kidney transplant recipients from six German transplant centres are randomised 2:1 to treatment with MIC (MIC group, N=42) or no treatment with MIC (control arm, N=21). MIC are manufactured from donor peripheral blood mononuclear cells under Good Manufacturing Practice conditions. The primary objective of this trial is to determine the efficacy of MIC treatment together with reduced conventional immunosuppressive therapy in terms of achieving an operational tolerance-like phenotype compared with SoC 12 months after MIC administration. Key secondary endpoints are the number of patient-relevant infections as well as a composite of biopsy-proven acute rejection, graft loss, graft dysfunction or death. Immunosuppressive therapy of MIC-treated patients is reduced during follow-up under an extended immunological monitoring including human leucocyte antigen-antibody testing, and determination of lymphocyte subsets, for example, regulatory B lymphocytes (Breg) and antidonor T cell response. A Data Safety Monitoring Board has been established to allow an independent assessment of safety and efficacy., Ethics and Dissemination: Ethical approval has been provided by the Ethics Committee of the Medical Faculty of the University of Heidelberg, Heidelberg, Germany (AFmu-580/2021, 17 March 2022) and from the Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Institute, Langen, Germany (Vorlage-Nr. 4586/02, 21 March 2022). Written informed consent will be obtained from all patients and respective donors prior to enrolment in the study. The results from the TOL-2 Study will be published in peer-reviewed medical journals and will be presented at symposia and scientific meetings., Trial Registration Number: NCT05365672., Competing Interests: Competing interests: CM, ASchmitt, MS, CK, GO, PT, MZ and MSchaier together with the University of Heidelberg, are cofounders of TolerogenixX GmbH, Heidelberg, Germany, a biotechnology company that holds licenses for MIC treatment. CK, GO, and PT hold a patent for MIC treatment. CM, ASchmitt, MSchmitt, CK, GO, CSüsal, PT, MZ, VD and MSchaier together with the University of Heidelberg and TolerogenixX GmbH filed a patent application for MIC treatment. JR is cofounder and shareholder of Trisaq, a biopharmaceutical company that develops novel therapy for kidney diseases., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background Venous thromboembolism (VTE) contributes significantly to COVID-19 morbidity and mortality. The urokinase receptor system is involved in the regulation of coagulation. Levels of soluble urokinase plasminogen activator receptor (suPAR) reflect hyperinflammation and are strongly predictive of outcomes in COVID-19. Whether suPAR levels identify patients with COVID-19 at risk for VTE is unclear. Methods and Results We leveraged a multinational observational study of patients hospitalized for COVID-19 with suPAR and D-dimer levels measured on admission. In 1960 patients (mean age, 58 years; 57% men; 20% Black race), we assessed the association between suPAR and incident VTE (defined as pulmonary embolism or deep vein thrombosis) using logistic regression and Fine-Gray modeling, accounting for the competing risk of death. VTE occurred in 163 (8%) patients and was associated with higher suPAR and D-dimer levels. There was a positive association between suPAR and D-dimer (β=7.34; P =0.002). Adjusted for clinical covariables, including D-dimer, the odds of VTE were 168% higher comparing the third with first suPAR tertiles (adjusted odds ratio, 2.68 [95% CI, 1.51-4.75]; P <0.001). Findings were consistent when stratified by D-dimer levels and in survival analysis accounting for death as a competing risk. On the basis of predicted probabilities from random forest, a decision tree found the combined D-dimer <1 mg/L and suPAR <11 ng/mL cutoffs, identifying 41% of patients with only 3.6% VTE probability. Conclusions Higher suPAR was associated with incident VTE independently of D-dimer in patients hospitalized for COVID-19. Combining suPAR and D-dimer identified patients at low VTE risk. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT04818866.

The objective of this study was to investigate the electroencephalographic reaction pattern and FOS protein expression in male piglets undergoing surgical castration under light isoflurane anaesthesia with or without local anaesthesia. The experiment was conducted under isoflurane anaesthesia to exclude the effect of the affective components of pain on the measurements. Changes in the oscillatory activity of the cerebral cortex over a 90 s period after noxious stimulation or simulated interventions were analysed. FOS expression was determined postmortem by performing immunohistochemistry in the dorsal horn of the spinal cord. The analysis of the response to an interdigital pinch revealed a biphasic reaction pattern in the electroencephalogram (EEG) that similarly was observed for the surgical stimuli during the castration procedure in the group without analgesia. This EEG response was attenuated or altered by the application of local anaesthetics. Immunohistochemical staining for FOS indicated a lower expression in the handling and in three local anaesthetic groups than in the animals castrated without pain relief. The findings indicate that EEG and FOS expression may serve as indicators for nociception in piglets under light isoflurane anaesthesia. A lower activation of nociceptive pathways occurs during castration after the application of local anaesthetics. However, EEG and FOS analyses should be combined with additional parameters to assess nociception, e.g., haemodynamic monitoring.

Caregivers are integral to the care of those with neurological disorders such as Parkinson's Disease (PD), but are often burdened by stress, anxiety, and depression. Previous research has suggested that the foundation of such stress is low-grade systemic inflammation, as evidenced by increased interleukin 6 (IL-6) and C-reactive protein (CRP) levels. Soluble urokinase-type plasminogen activator receptor (suPAR) is a kidney disease risk factor and marker of chronic inflammation that integrates psycho-social stress and organ dysfunction. Caregivers of PD experience an extraordinary amount of stress and suPAR's role as prognostic marker has not yet been assessed in caregivers of PD. The aim of this study was to determine the relationship between suPAR levels and PD caregiver burden. Healthy volunteers who accompanied patients with parkinsonism (n = 35) donated blood samples, and complete blood counts (CBC), CRP, and suPAR levels were measured. Participants were then interviewed by telephone and stratified into primary and non-primary caregiver groups. Their caregiver burden was quantified through the Zarit Caregiver Burden Short Form (ZBI-12). The resultant data demonstrated higher plasma levels of suPAR and ZBI-12 scores for the primary caregiver group relative to the non-primary caregiver group (suPAR level: 3.73 vs. 2.72 ng/mL, p = 0.01; ZBI-12: 18.57 vs. 5.4, p < 0.0001; Table). The data also revealed a moderate positive correlation between suPAR and ZBI-12 scores. These findings not only demonstrate a correlation between elevated suPAR and caregiving burden in PD, but also further support and raise awareness for the overall psychosocial burden and stress experienced by those caregivers., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Inflammation is a common feature of many kidney diseases. The implicated inflammatory mediators and their underlying molecular mechanisms however are often not clear., Summary: suPAR is the soluble form of urokinase-type plasminogen activator receptor (uPAR), associated with inflammation and immune activation. It has evolved into a unique circulating kidney disease factor over the last 10 years. In particular, suPAR has multiple looks due to enzymatic cleavage and alternative transcriptional splicing of the uPAR gene. Most recently, suPAR has emerged as a systemic mediator for COVID-19 infection, associated with lung as well as kidney dysfunction. Like membrane-bound uPAR, suPAR could interact with integrins (e.g., αvβ3 integrin) on podocytes, providing the molecular basis for some glomerular kidney diseases. In addition, there have been numerous studies suggesting that suPAR connects acute kidney injury to chronic kidney disease as a special kidney risk factor. Moreover, the implication of circulating suPAR levels in kidney transplantation and plasmapheresis not only indicates its relevance in monitoring for recurrence but also implies suPAR as a possible therapeutic target. In fact, the therapeutic concept of manipulating suPAR function has been evidenced in several kidney disease experimental models., Key Messages: The last 10 years of research has established suPAR as a unique inflammatory mediator for kidneys. While open questions remain and deserve additional studies, modulating suPAR function may represent a promising novel therapeutic strategy for kidney disease., Competing Interests: J.R. reports personal fees from Biomarin, Visterra, Astellas, Genentech, Merck, Gerson Lehrman Group, and Massachusetts General Hospital. He is the recipient of grants from Nephcure Kidney International and Thermo BCT. J.R.'s lab is the recipient of fee-for-service funds from Walden Biosciences. J.R. is cofounder, scientific advisory board co-chair, and shareholder of Walden Biosciences, a kidney therapeutic company. Other 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 by S. Karger AG, Basel.)