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Abstract Circulating leukocytes enter tissue either through endothelial junctions (paracellular) or via a pore through the body of endothelial cells (transcellular). We have previously shown that genetically replacing VE-cadherin with a VE-cadherin-α-catenin (VEC-αC) fusion construct—which binds constitutively to actin—obstructs junctions, and blocks leukocyte extravasation in lung, skin and postcapillary venules of cremaster muscle. However, neutrophil recruitment into the inflamed peritoneal cavity was unimpaired. Investigating reasons for this, here, we visualized neutrophil diapedesis by 3D intravital video microscopy in the cremaster muscle and omentum, the major site of neutrophil recruitment into the peritoneal cavity. We found that 80% of neutrophil-extravasation occurred through HEVs in the omentum, which was unimpaired by VEC-αC. In addition, in larger venules (60–85 µm) of both tissues, less than 15% of neutrophils extravasated transcellularly in WT mice. However, in VEC-α-C mice, transcellular diapedesis increased severalfold in the omentum, but not in the cremaster. In line with this, omental venules expressed higher levels of ICAM-1 and atypical chemokine receptor 1. Furthermore, only in the omentum, VEC-αC expression caused reduced elongation of venular endothelium in flow-direction, suggesting different biomechanical properties. Collectively, VEC-αC does not inhibit paracellular transmigration in all types of venules and can modulate the diapedesis route.

Abstract Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.

ObjectiveTo evaluate the association between different types of human milk feeds and bronchopulmonary dysplasia (BPD) in preterm infants.MethodsData on dispensed mother’s own milk (MOM) and donor human milk (DHM) from Leipzig Milk Bank for hospitalized infants with a gestational age (GA) ≤32 weeks observed from birth to 36 weeks’ postmenstrual age or prior discharge were used. BPD was assessed based on documented International Statistical Classification of Diseases and Related Health Problems (ICD) diagnosis and on electronic hospital records (EHR) of data on ventilation and oxygen supplementation. Associations of dispensed milk feed variations with BPD were investigated using logistic regressions in crude and adjusted models.Results866 infants were included with a BPD prevalence of 15.4% (EHR) and 23.2% (ICD). The mean GA was 29.1 weeks. The majority (84.4%, n = 746) of infants were nurtured with a mix of MOM, DHM supplemented by formula or parenteral (other) nutrition during hospitalization. For which, MOM comprised the highest median [Q1–Q3] percentage proportion (53[31–81] %) of this mix. Exclusive fresh milk and exclusive MOM feeds were dispensed on a mean of 40 and 34% patient-days, respectively. Statistically significant associations with lower BPD incidence were only observed for 70–80% MOM vs. DHM, and 60% fresh vs. frozen milk, in crude and adjusted models.ConclusionOur findings suggest a protective association of MOM and fresh milk with lower odds of BPD, which may be dependent on the proportion of MOM or fresh milk administered. These results highlight the importance of MOM as an ideal source of nutrition during early infancy.

Abstract Objectives The objective of this study is to assess frequency and risk factors for intraoperative hypoxemia of the lower limbs during robot‐assisted radical prostatectomy (RARP). Trendelenburg position during RARP may contribute to hypoxemia and compartment syndrome (CS) of the lower limbs as a major but rare complication. Patients and methods This prospective study included patients undergoing RARP for prostate cancer. Preoperative calculation of the ankle‐brachial‐index (ABI) was performed. Peripheral oxygen saturation (SpO2) at the toes was routinely measured. Occurrence of SpO2 levels of

Abstract Background Autoimmune hepatitis (AIH) is a rare, chronic inflammatory disease of the liver. The treatment goal is reaching complete biochemical response (CR), defined as the normalisation of aspartate and alanine aminotransferases and immunoglobulin gamma. Ongoing AIH activity can lead to fibrosis and (decompensated) cirrhosis. Incomplete biochemical response is the most important risk factor for liver transplantation or liver-related mortality. First-line treatment consists of a combination of azathioprine and prednisolone. If CR is not reached, tacrolimus (TAC) or mycophenolate mofetil (MMF) can be used as second-line therapy. Both products are registered for the prevention of graft rejection in solid organ transplant recipients. The aim of this study is to compare the effectiveness and safety of TAC and MMF as second-line treatment for AIH. Methods The TAILOR study is a phase IIIB, multicentre, open-label, parallel-group, randomised (1:1) controlled trial performed in large teaching and university hospitals in the Netherlands. We will enrol 86 patients with AIH who have not reached CR after at least 6 months of treatment with first-line therapy. Patients are randomised to TAC (0.07 mg/kg/day initially and adjusted by trough levels) or MMF (max 2000 mg/day), stratified by the presence of cirrhosis at inclusion. The primary endpoint is the difference in the proportion of patients reaching CR after 12 months. Secondary endpoints include the difference in the proportion of patients reaching CR after 6 months, adverse effects, difference in fibrogenesis, quality of life and cost-effectiveness. Discussion This is the first randomised controlled trial comparing two second-line therapies for AIH. Currently, second-line treatment is based on retrospective cohort studies. The rarity of AIH is the main issue in clinical research for alternative treatment options. The results of this trial can be implemented in existing international clinical guidelines. Trial registration ClinicalTrials.gov NCT05221411 . Retrospectively registered on 3 February 2022; EudraCT number 2021–003420-33. Prospectively registered on 16 June 2021.

Abstract Acetylation could improve the bioavailability of (-)-Epigallocatechin-3-Gallate (EGCG), but the relationship of substitution degree and antioxidant capacity of acetylated EGCG was unclear. The acetylated EGCG products were separated by preparation high performance liquid chromatography (HPLC). Two mono substituted acetylated EGCG, three substituted acetylated EGCG (T-AcE), eight substituted acetylated EGCG (E-AcE) and (-)-Epigallocatechin gallate (EGCG) were isolated. The 7-acetyl-EGCG (S7-ACEGCG) and 7-acetyl-EGCG (T-AcE) were identified for the first time. The antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities of EGCG decreased significantly after acetylation modification. The more EGCG acetylation modification sites, the lower the total antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities. The antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities of 5-acetyl-EGCG (S5-ACE) were higher than 7-acetyl-EGCG (S7-AcE). Combining all the results in this and previous studies, acetylation modification is not conducive to the performance of EGCG antioxidant capacity.

Remote ischemic preconditioning (RIPC) reduces ischemia-reperfusion injury in aortocoronary bypass surgery, potentially via extracellular vesicles (EVs) and their micro-RNA content. Clinical data implicate that propofol might inhibit the cardioprotective RIPC effect. This prospective, randomized study investigated the influence of different anesthetic regimes on RIPC efficacy and EV micro-RNA signatures. We also assessed the impact of propofol on cell protection after hypoxic conditioning and EV-mediated RIPC in vitro. H9c2 rat cardiomyoblasts were subjected to hypoxia, with or without propofol, and subsequent simulated ischemia-reperfusion injury. Apoptosis was measured by flow cytometry. Blood samples of 64 patients receiving anesthetic maintenance with propofol or isoflurane, along with RIPC or sham procedures, were analyzed, and EVs were enriched using a polymer-based method. Propofol administration corresponded with increased Troponin T levels (4669 ± 435.6 pg/mL), suggesting an inhibition of the cardioprotective RIPC effect. RIPC leads to a notable rise in miR-21 concentrations in the group receiving propofol anesthesia (fold change 7.22 ± 6.6). In vitro experiments showed that apoptosis reduction was compromised with propofol and only occurred in an EV-enriched preconditioning medium, not in an EV-depleted medium. Our study could clinically and experimentally confirm propofol inhibition of RIPC protection. Increased miR-21 expression could provide evidence for a possible inhibitory mechanism.

Thymic epithelial cells are indispensable for T cell maturation and selection and the induction of central immune tolerance. The self-peptide repertoire expressed by medullary thymic epithelial cells is in part regulated by the transcriptional regulator Aire (Autoimmune regulator) and the transcription factor Fezf2. Due to the high complexity of mTEC maturation stages (i.e., post-Aire, Krt10+ mTECs, and Dclk1+ Tuft mTECs) and the heterogeneity in their gene expression profiles (i.e., mosaic expression patterns), it has been challenging to identify the additional factors complementing the transcriptional regulation. We aimed to identify the transcriptional regulators involved in the regulation of mTEC development and self-peptide expression in an unbiased and genome-wide manner. We used ATAC footprinting analysis as an indirect approach to identify transcription factors involved in the gene expression regulation in mTECs, which we validated by ChIP sequencing. This study identifies Fezf2 as a regulator of the recently described thymic Tuft cells (i.e., Tuft mTECs). Furthermore, we identify that transcriptional regulators of the ELF, ESE, ERF, and PEA3 subfamily of the ETS transcription factor family and members of the Krüppel-like family of transcription factors play a role in the transcriptional regulation of genes involved in late mTEC development and promiscuous gene expression.

IntroductionAn increasing amount of longitudinal health data is available on critically ill septic patients in the age of digital medicine, including daily sequential organ failure assessment (SOFA) score measurements. Thus, the assessment in sepsis focuses increasingly on the evaluation of the individual disease's trajectory. Machine learning (ML) algorithms may provide a promising approach here to improve the evaluation of daily SOFA score dynamics. We tested whether ML algorithms can outperform the conventional ΔSOFA score regarding the accuracy of 30-day mortality prediction.MethodsWe used the multicentric SepsisDataNet.NRW study cohort that prospectively enrolled 252 sepsis patients between 03/2018 and 09/2019 for training ML algorithms, i.e. support vector machine (SVM) with polynomial kernel and artificial neural network (aNN). We used the Amsterdam UMC database covering 1,790 sepsis patients for external and independent validation.ResultsBoth SVM (AUC 0.84; 95% CI: 0.71-0.96) and aNN (AUC 0.82; 95% CI: 0.69-0.95) assessing the SOFA scores of the first seven days led to a more accurate prognosis of 30-day mortality compared to the ΔSOFA score between day 1 and 7 (AUC 0.73; 95% CI: 0.65-0.80; p = 0.02 and p = 0.05, respectively). These differences were even more prominent the shorter the time interval considered. Using the SOFA scores of day 1 to 3 SVM (AUC 0.82; 95% CI: 0.68 0.95) and aNN (AUC 0.80; 95% CI: 0.660.93) led to a more accurate prognosis of 30-day mortality compared to the ΔSOFA score (AUC 0.66; 95% CI: 0.58-0.74; p < 0.01 and p < 0.01, respectively). Strikingly, all these findings could be confirmed in the independent external validation cohort.ConclusionsThe ML-based algorithms using daily SOFA scores markedly improved the accuracy of mortality compared to the conventional ΔSOFA score. Therefore, this approach could provide a promising and automated approach to assess the individual disease trajectory in sepsis. These findings reflect the potential of incorporating ML algorithms as robust and generalizable support tools on intensive care units.

Summary: Fleas transmit Yersinia pestis directly within the dermis of mammals to cause bubonic plague. Syringe-mediated inoculation is widely used to recapitulate bubonic plague and study Y. pestis pathogenesis. However, intradermal needle inoculation is tedious, error prone, and poses a significant safety risk for laboratorians. Microneedle arrays (MNAs) are micron-scale polymeric structures that deliver materials to the dermis, while minimizing the risk of needle sticks. We demonstrated that MNA inoculation is a viable strategy to recapitulate bubonic plague and study bacterial virulence by defining the parameters needed to establish a lethal infection in the mouse model and characterizing the course of infection using live-animal optical imaging. Using MNAs, we also demonstrated that Y. pestis must overcome calprotectin-mediated zinc restriction within the dermis and dermal delivery of an attenuated mutant has vaccine potential. Together, these data demonstrate that MNAs are a safe alternative to study Y. pestis pathogenesis in the laboratory.

Introduction: Regulatory T cells (Tregs) play a critical role in the maintenance of immune homeostasis but also protect tumors from immune-mediated growth control or rejection and pose a significant barrier to effective immunotherapy. Inhibition of MALT1 paracaspase activity can selectively reprogram immune-suppressive Tregs in the tumor microenvironment to adopt a proinflammatory fragile state, which offers an opportunity to impede tumor growth and enhance the efficacy of immune checkpoint therapy (ICT). Methods: We performed preclinical studies with the orally available allosteric MALT1 inhibitor (S)-mepazine as a single-agent and in combination with anti-programmed cell death protein 1 (PD-1) ICT to investigate its pharmacokinetic properties and antitumor effects in several murine tumor models as well as patient-derived organotypic tumor spheroids (PDOTS). Results: (S)-mepazine demonstrated significant antitumor effects and was synergistic with anti-PD-1 therapy in vivo and ex vivo but did not affect circulating Treg frequencies in healthy rats at effective doses. Pharmacokinetic profiling revealed favorable drug accumulation in tumors to concentrations that effectively blocked MALT1 activity, potentially explaining preferential effects on tumor-infiltrating over systemic Tregs. Conclusions: The MALT1 inhibitor (S)-mepazine showed single-agent anticancer activity and presents a promising opportunity for combination with PD-1 pathway-targeted ICT. Activity in syngeneic tumor models and human PDOTS was likely mediated by induction of tumor-associated Treg fragility. This translational study supports ongoing clinical investigations (ClinicalTrials.gov Identifier: NCT04859777) of MPT-0118, (S)-mepazine succinate, in patients with advanced or metastatic treatment-refractory solid tumors.

Abstract The use of extracorporeal membrane oxygenation (ECMO) is discussed to improve patients’ outcome in severe COVID-19 with respiratory failure, but data on ECMO remains controversial. The aim of the study was to determine the characteristics of patients under invasive mechanical ventilation (IMV) with or without veno-venous ECMO support and to evaluate outcome parameters. Ventilated patients with COVID-19 with and without additional ECMO support were analyzed in a retrospective multicenter study regarding clinical characteristics, respiratory and laboratory parameters in day-to-day follow-up. Recruitment of patients was conducted during the first three COVID-19 waves at four German university hospitals of the Ruhr University Bochum, located in the Middle Ruhr Region. From March 1, 2020 to August 31, 2021, the charts of 149 patients who were ventilated for COVID-19 infection, were included (63.8% male, median age 67 years). Fifty patients (33.6%) received additional ECMO support. On average, ECMO therapy was initiated 15.6 ± 9.4 days after symptom onset, 10.6 ± 7.1 days after hospital admission, and 4.8 ± 6.4 days after the start of IMV. Male sex and higher SOFA and RESP scores were observed significantly more often in the high-volume ECMO center. Pre-medication with antidepressants was more often detected in survivors (22.0% vs. 6.5%; p = 0.006). ECMO patients were 14 years younger and presented a lower rate of concomitant cardiovascular diseases (18.0% vs. 47.5%; p = 0.0004). Additionally, cytokine-adsorption (46.0% vs. 13.1%; p

BackgroundHuman milk banking has become an important aspect of Nutritional medicine. It is not just about the provision of mother’s own milk (MOM) or donor human milk (DHM) in the hospital, but also a strategy to encourage breastfeeding in the clinical setting and beyond.ObjectiveTo describe the feeding patterns of hospitalised infants including human milk dispensed by the Leipzig Donor Human Milk Bank (LMB).DesignA descriptive analysis of daily data on milk feeds dispensed by LMB for hospitalised infants distinguishing between MOM or DHM, either fresh or frozen, and raw/pasteurised milk from 2012–2019.ResultsWe included 2,562 infants with median hospitalisation of 23 days, for whom human milk was dispensed on median 76% of those days and other nutrition on the remaining days. Raw MOM and raw DHM comprised 52% and 8% of the dispensed milk, respectively. Dispensing exclusive DHM instead of MOM for at least one full day was required for 55% of the infants, mostly at the beginning but also later during hospitalisation. Exclusive raw DHM was dispensed on at least 1 day for 37% of the infants, in different birthweight strata 2,500 g: 3%. At discharge, MOM was dispensed for more than 60% of the infants.ConclusionDuring an infant’s hospital stay, LMB dispenses various human milk feeds with interspersed DHM resulting in complex intra-individual and time-variant feeding patterns. LMB dispenses raw MOM and especially raw DHM with the intention to retain the properties of human milk unlike a diet containing pasteurised DHM and/or formula. Although raw DHM comprises a small percentage of all dispensed milk, raw DHM is dispensed for a substantial portion of infants. Our results document that dispensing raw DHM, is possible in routine settings.

BackgroundRandomized controlled trials have indicated reduced mortality rates in very preterm infants assigned to high compared to low oxygen saturation (SpO2) target levels, accompanied by higher rates of retinopathy of prematurity and bronchopulmonary dysplasia. However, the benefit-to-harm ratio may depend on the local background mortality risk. We therefore aimed to quantify the risk–benefit ratios of different SpO2 target ranges in 10 tertiary newborn intensive care units (NICUs) in East Germany.MethodsIn a retrospective multicenter study, 1,399 infants born between 2008 and 2012 at a gestational age between 24 0/7 and 27 6/7 weeks and with a birthweight below 1,250 g were grouped according to the hospital's target SpO2 range [high oxygen saturation group (HOSG) above 90%], low oxygen saturation group (LOSG) below 90%] and the compliance of units with their target SpO2 range. The association between neonatal morbidities, neurodevelopmental outcomes, selected treatment strategies, and target SpO2 ranges was calculated using chi-squared and Mann Whitney U tests.ResultsNine of the ten participating NICUs met their SpO2 target ranges. Five units were considered as HOSG, and five units were considered as LOSG. Necrotizing enterocolitis and intraventricular hemorrhage grade ≥ 2 occurred significantly more frequently in the HOSG than in the LOSG (8.4% vs. 5.1%, p = 0.02; and 26.6% vs. 17.7%, p

Abstract Background Echocardiographic quantification of ejection fraction (EF) by manual endocardial tracing requires training, is time-consuming and potentially user-dependent, whereas determination of cardiac output by pulmonary artery catheterization (PAC) is invasive and carries a risk of complications. Recently, a novel software for semi-automated EF and CO assessment (AutoEF) using transthoracic echocardiography (TTE) has been introduced. We hypothesized that AutoEF would provide EF values different from those obtained by the modified Simpson’s method in transoesophageal echocardiography (TOE) and that AutoEF CO measurements would not agree with those obtained via VTILVOT in TOE and by thermodilution using PAC. Methods In 167 patients undergoing coronary artery bypass graft surgery (CABG), TTE cine loops of apical 4- and 2-chamber views were recorded after anaesthesia induction under steady-state conditions. Subsequently, TOE was performed following a standardized protocol, and CO was determined by thermodilution. EF and CO were assessed by TTE AutoEF as well as TOE, using the modified Simpson’s method, and Doppler measurements via velocity time integral in the LV outflow tract (VTILVOT). We determined Pearson’s correlation coefficients r and carried out Bland–Altman analyses. The primary endpoints were differences in EF and CO. The secondary endpoints were differences in left ventricular volumes at end diastole (LVEDV) and end systole (LVESV). Results AutoEF and the modified Simpson’s method in TOE showed moderate EF correlation (r = 0.38, p

Abstract Background Currently, the standard therapy for autoimmune hepatitis (AIH) consists of a combination of prednisolone and azathioprine. However, 15% of patients are intolerant to azathioprine which necessitates cessation of azathioprine or changes in therapy. In addition, not all patients achieve complete biochemical response (CR). Uncontrolled data indicate that mycophenolate mofetil (MMF) can induce CR in a majority of patients. Better understanding of first-line treatment and robust evidence from randomised clinical trials are needed. The aim of this study was to explore the potential benefits of MMF as compared to azathioprine, both combined with prednisolone, as induction therapy in a randomised controlled trial in patients with treatment-naive AIH. Methods CAMARO is a randomised (1:1), open-label, parallel-group, multicentre superiority trial. All patients with AIH are screened for eligibility. Seventy adult patients with AIH from fourteen centres in the Netherlands and Belgium will be randomised to receive MMF or azathioprine. Both treatment arms will start with prednisolone as induction therapy. The primary outcome is biochemical remission, defined as serum levels of alanine aminotransferase and immunoglobulin G below the upper limit of normal. Secondary outcomes include safety and tolerability of MMF and azathioprine, time to remission, changes in Model For End-Stage Liver Disease (MELD)-score, adverse events, and aspects of quality of life. The study period will last for 24 weeks. Discussion The CAMARO trial investigates whether treatment with MMF and prednisolone increases the proportion of patients in remission compared with azathioprine and prednisolone as the current standard treatment strategy. In addition, we reflect on the challenges of conducting a randomized trial in rare diseases. Trial registration EudraCT 2016-001038-91 . Prospectively registered on 18 April 2016. Graphical Abstract

Background: Although there are several efficacious vaccines against COVID-19, vaccination rates in many regions around the world remain insufficient to prevent continued high disease burden and emergence of viral variants. Repurposing of existing therapeutics that prevent or mitigate severe COVID-19 could help to address these challenges. The objective of this study was to determine whether prior use of bisphosphonates is associated with reduced incidence and/or severity of COVID-19. Methods: A retrospective cohort study utilizing payer-complete health insurance claims data from 8,239,790 patients with continuous medical and prescription insurance January 1, 2019 to June 30, 2020 was performed. The primary exposure of interest was use of any bisphosphonate from January 1, 2019 to February 29, 2020. Bisphosphonate users were identified as patients having at least one bisphosphonate claim during this period, who were then 1:1 propensity score-matched to bisphosphonate non-users by age, gender, insurance type, primary-care-provider visit in 2019, and comorbidity burden. Main outcomes of interest included: (a) any testing for SARS-CoV-2 infection; (b) COVID-19 diagnosis; and (c) hospitalization with a COVID-19 diagnosis between March 1, 2020 and June 30, 2020. Multiple sensitivity analyses were also performed to assess core study outcomes amongst more restrictive matches between BP users/non-users, as well as assessing the relationship between BP-use and other respiratory infections (pneumonia, acute bronchitis) both during the same study period as well as before the COVID outbreak. Results: A total of 7,906,603 patients for whom continuous medical and prescription insurance information was available were selected. A total of 450,366 bisphosphonate users were identified and 1:1 propensity score-matched to bisphosphonate non-users. Bisphosphonate users had lower odds ratios (OR) of testing for SARS-CoV-2 infection (OR = 0.22; 95%CI:0.21–0.23; p