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Abstract Tuberculosis (TB) remains one of the top 10 causes of death worldwide and still poses a serious challenge to public health. Recent attention to neutrophils has uncovered unexplored areas demanding further investigation. Therefore, the aim of this study was to determine neutrophil activation and circulatory neutrophil extracellular trap (NET) formation in various types of TB. Sera from TB patients (n = 91) and healthy controls (NHD; n = 38) were analyzed for NE-DNA and MPO–DNA complexes, cell-free DNA (cfDNA), and protease activity (elastase). We show that these NET parameters were increased in TB sera. Importantly, NET formation and NE activity were elevated in TB patients with extensive tissue damage when compared to those with minor damage and in patients with relapse, compared to new cases. We discuss the importance of balancing NET formation to prevent tissue damage or even relapse and argue to analyze circulating NET parameters to monitor the risk of disease relapse. To investigate the tissues for NETs and to find the source of the circulating NET degradation products, we collected sections of granulomas in lung and lymph node biopsies. Samples from other diseases with granulomas, including sarcoidosis (SARC) and apical periodontitis (AP), served as controls. Whereas NET formation characterizes the caseating granulomas, both caseating and non-caseating granulomas harbor DNA with unusual conformation. As TB is associated with hypercoagulation and thromboembolism, we further imaged the pulmonary vessels of TB patients and detected vascular occlusions with neutrophil aggregates. This highlights the dual role of neutrophils in the pathology of TB.

Worldwide, 150 children are born each day with congenital diaphragmatic hernia (CDH), a diaphragmatic defect with concomitant abnormal lung development. Patients with CDH with large defects are particularly challenging to treat, have the highest mortality, and are at significant risk of long-term complications. Advances in prenatal and neonatal treatments have improved survival in high-risk patients with CDH, but surgical treatment of large defects lacks standardization. Open repair by an abdominal approach has long been considered the traditional procedure, but the type of defect repair (patch or muscle flap) and patch material (non-absorbable, synthetic or absorbable, biological) remain subjects of debate. Increased experience and improved techniques in minimally invasive surgery (MIS) have expanded selection criteria for thoracoscopic defect repair in cardiopulmonary stable patients with small defects. However, the application of MIS to repair large defects remains controversial due to increased recurrence rates and unknown long-term effects of perioperative hypercapnia and acidosis resulting from capnothorax and reduced ventilation. Current recommendations on the surgical management rely on cohort studies of varying patient numbers and data on the long-term outcomes are sparse. Here, we discuss surgical approaches for diaphragmatic defect repair highlighting advancements, and knowledge gaps in surgical techniques (open surgery and MIS), patch materials and muscle flaps for large defects, as well as procedural adjuncts and management of CDH variants.

Necrotizing enterocolitis (NEC) is one of the most devasting diseases affecting preterm neonates. However, despite a lot of research, NEC’s pathogenesis remains unclear. It is known that the pathogenesis is a multifactorial process, including (1) a pathological microbiome with abnormal bacterial colonization, (2) an immature immune system, (3) enteral feeding, (3) an impairment of microcirculation, and (4) possibly ischemia-reperfusion damage to the intestine. Overall, the immaturity of the mucosal barrier and the increased expression of Toll-like receptor 4 (TLR4) within the intestinal epithelium result in an intestinal hyperinflammation reaction. Concurrently, a deficiency in counter-regulatory mediators can be seen. The sum of these processes can ultimately result in intestinal necrosis leading to very high mortality rates of the affected neonates. In the last decade no substantial advances in the treatment of NEC have been made. Thus, NEC animal models as well as in vitro models have been employed to better understand NEC’s pathogenesis on a cellular and molecular level. This review will highlight the different models currently in use to study immunological aspects of NEC.

Chimeric antigen receptor (CAR) T cell therapy has effectively complemented the treatment of advanced relapsed and refractory hematological cancers. The remarkable achievements of CD19- and BCMA-CAR T therapies have raised high expectations within the fields of hematology and oncology. These groundbreaking successes are propelling a collective aspiration to extend the reach of CAR therapies beyond B-lineage malignancies. Advanced CAR technologies have created a momentum to surmount the limitations of conventional CAR concepts. Most importantly, innovations that enable combinatorial targeting to address target antigen heterogeneity, using versatile adapter CAR concepts in conjunction with recent transformative next-generation CAR design, offer the promise to overcome both the bottleneck associated with CAR manufacturing and patient-individualized treatment regimens. In this comprehensive review, we delineate the fundamental prerequisites, navigate through pivotal challenges, and elucidate strategic approaches, all aimed at paving the way for the future establishment of multitargeted immunotherapies using universal CAR technologies.

Hirschsprung’s disease (HSCR, incidence 1/5000 live births) is caused by the failure of neural crest-derived precursors to migrate, survive, proliferate, or differentiate during the embryonic development of the Enteric Nervous System (ENS), which could be disrupted by many factors, including inflammatory processes. The NF-κB family controls several biological processes, including inflammation, neurogenesis, and cell migration. With the aim of studying the potential role of NF-κB in HSCR, we have analyzed the expression of the NF-κB main subunits and other NF-κB-related genes by RT-qPCR in HSCR tissue samples (sub-divided into ganglionic and aganglionic segments). We found decreased gene expression of the NF-κB main subunit RELA but also of NFKBIA, TNFA, TFGBR2, and ERBB3 in the pathologic distal aganglionic segments compared to the proximal ganglionic segments. Moreover, we could also confirm the lower protein expression of RelA/p65 in the aganglionic distal segments by immunofluorescence staining. Further, we show that the expression of RelA/p65 protein in the proximal segments concurs with lymphocyte infiltration in the bowel tissue, indicating a pro-inflammatory activation of p65 in the proximal ganglionic HSCR tissue in the patients analyzed. All in all, our findings suggest that the modulation of NF-κB signaling in the neuro-enteric system does obviously contribute to the pathological effects of HSCR.

Background: Abdominal tumors rarely occur in childhood but are associated with great psychological stress for patients and their families. Parents playing a central role in their children’s treatment may experience adverse effects on their Health-Related Quality of Life (HRQoL) and mental health due to the children’s illness and required treatment. Given the limited knowledge of the psychosocial burden of parents with children suffering from solid abdominal tumors, this study aims to assess their HRQoL and mental health along with the impact of various psychosocial factors. Methods: A cross-sectional cohort study was carried out involving 54 parents of children with solid abdominal tumors who had surgery at the University Medical Center Hamburg-Eppendorf in Germany. The results were compared with 46 parents of children undergoing routine surgeries without a prior tumor diagnosis, and with normative values. Data were obtained using standardized questionnaires. Results: Parents of the index group showed significantly worse HRQoL and limited mental health. Furthermore, they showed significantly higher parental burden in several subscales and less social support in comparison with the control group. Conclusions: Considering the lower parental HRQoL and the central role parents play in their children’s lives, it is crucial to include prevention and intervention measures for parents as part of a multimodal treatment regime for children with solid pediatric abdominal tumors.

Post-surgical abdominal adhesions, although poorly understood, are highly prevalent. The molecular processes underlying their formation remain elusive. This review aims to assess the relationship between neutrophil extracellular traps (NETs) and the generation of postoperative peritoneal adhesions and to discuss methods for mitigating peritoneal adhesions. A keyword or medical subject heading (MeSH) search for all original articles and reviews was performed in PubMed and Google Scholar. It included studies assessing peritoneal adhesion reformation after abdominal surgery from 2003 to 2023. After assessing for eligibility, the selected articles were evaluated using the Critical Appraisal Skills Programme checklist for qualitative research. The search yielded 127 full-text articles for assessment of eligibility, of which 7 studies met our criteria and were subjected to a detailed quality review using the Critical Appraisal Skills Programme (CASP) checklist. The selected studies offer a comprehensive analysis of adhesion pathogenesis with a special focus on the role of neutrophil extracellular traps (NETs) in the development of peritoneal adhesions. Current interventional strategies are examined, including the use of mechanical barriers, advances in regenerative medicine, and targeted molecular therapies. In particular, this review emphasizes the potential of NET-targeted interventions as promising strategies to mitigate postoperative adhesion development. Evidence suggests that in addition to their role in innate defense against infections and autoimmune diseases, NETs also play a crucial role in the formation of peritoneal adhesions after surgery. Therefore, therapeutic strategies that target NETs are emerging as significant considerations for researchers. Continued research is vital to fully elucidate the relationship between NETs and post-surgical adhesion formation to develop effective treatments.

Abstract Pectus excavatum (PE) is a congenital malformation with a funnel-shaped depression of the sternum that can lead to cardiac symptoms. However, there are patients with thoracic constriction (defined as elevated Haller-Index > 3.25 determined by cardiac magnetic resonance imaging (CMR)) without visible evidence of PE, leading to similar complaints. Between January 2004 till June 2020, patients who underwent CMR for further evaluation of the heart, due to cardiac symptoms were enrolled and compared to controls. Biventricular global strain analysis was assessed using feature tracking (CMR-FT). ECG and/or Holter recordings were performed to detect rhythm events. Cardiac symptoms were evaluated in detail using a questionnaire. Finally, 88 patients (male 35, female 53) with elevated Haller-Index (3.9 ± 0.8) were included and compared to CMR data from 25 individuals with confirmed PE and 25 healthy controls (HC). Mean age at time of CMR was 35 ± 16 years. The most common symptoms at presentation were palpitations (41%), followed by dyspnea (24%) and atypical chest pain (14%). Three patients (3%) had atrial fibrillation or atrial flutter. Concomitant phenomena were pericardial effusion in 39% and mitral valve prolapse (MVP) in 27% of the study cohort. While there were no differences in left ventricular function or volumes, right ventricular function (RVEF) was significantly lower in patients with internal PE compared to HC (RVEF (%) 50 ± 5 vs 59 ± 4, p

IntroductionNeutrophil extracellular traps (NETs) have various beneficial and detrimental effects in the body. It has been reported that some bacteria may evade the immune system when entangled in NETs. Thus, the aim of the current study was to evaluate the effects of a combined DNase and antibiotic therapy in a murine model of abdominal sepsis.MethodsC57BL/6 mice underwent a cecum-ligation-and-puncture procedure. We used wild-type and knockout mice with the same genetic background (PAD4-KO and DNase1-KO). Mice were treated with (I) antibiotics (Metronidazol/Cefuroxime), (II) DNAse1, or (III) with the combination of both; mock-treated mice served as controls. We employed a streak plate procedure and 16s-RNA analysis to evaluate bacterial translocation and quantified NETs formation by ELISA and immune fluorescence. Western blot and proteomics analysis were used to determine inflammation.ResultsA total of n=73 mice were used. Mice that were genetically unable to produce extended NETs or were treated with DNases displayed superior survival and bacterial clearance and reduced inflammation. DNase1 treatment significantly improved clearance of Gram-negative bacteria and survival rates. Importantly, the combination of DNase1 and antibiotics reduced tissue damage, neutrophil activation, and NETs formation in the affected intestinal tissue.ConclusionThe combination of antibiotics with DNase1 ameliorates abdominal sepsis. Gram-negative bacteria are cleared better when NETs are cleaved by DNase1. Future studies on antibiotic therapy should be combined with anti-NETs therapies.

Summary: Peritoneal adhesions are poorly understood but highly prevalent conditions that can cause intestinal obstruction and pelvic pain requiring surgery. While there is consensus that stress-induced inflammation triggers peritoneal adhesions, the molecular processes of their formation still remain elusive. We performed murine models and analyzed human samples to monitor the formation of adhesions and the treatment with DNases. Various molecular analyses were used to evaluate the adhesions. The experimental peritoneal adhesions of the murine models and biopsy material from humans are largely based on neutrophil extracellular traps (NETs). Treatment with DNASE1 (Dornase alfa) and the human DNASE1L3 analog (NTR-10), significantly reduced peritoneal adhesions in experimental models.We conclude that NETs serve as essential scaffold for the formation of adhesions; DNases interfere with this process. Herein, we show that therapeutic application of DNases can be employed to prevent the formation of murine peritoneal adhesions. If this can be translated into the human situation requires clinical studies.

Despite limited research, existing studies using generic quality of life (QOL) tools indicate decreased physical health and compromised emotional functioning in children with IF. This study investigates QOL in children with short bowel syndrome (SBS) and its determinants. The study included 57 pediatric patients with SBS treated at Mannheim’s University Hospital between 1998 and 2014. To evaluate QOL, the KINDL questionnaire was used. Three age-specific questionnaire variants were employed, and parental proxy reports were collected. Most patients underwent intestinal lengthening procedures, with varying primary diagnoses. A comparison with healthy children from the patient’s perspective revealed no difference but from the parent’s perspective showed lower QOL in SBS patients, especially regarding physical and mental well-being. QOL varied with age, with 7–10-year-olds reporting the lowest scores. Several factors, including independence from parenteral nutrition and the presence of a complete colon, positively influenced QOL. The independence of parenteral nutrition and the presence of a complete colon positively influenced QOL. The Bianchi technique for intestinal lengthening has also shown promise but needs further research. The observation sample in this study is too small to generalize about the whole population of SBS patients. However, this study shows that many health and treatment factors affect QOL, and a large multicenter study is necessary. Our findings underline the importance of appropriate psychological support for children with SBS and their families.

This comprehensive review examines the role of Neutrophil Extracellular Traps (NETs) in pediatric surgery. Focusing on NET formation, functions, and implications, this study highlights their dual impact in infection control and contribution to tissue damage after surgery. It covers the role of NET formation in a range of pediatric conditions including immunothrombosis, formation of peritoneal adhesions, appendicitis, burns, gallstones, tumors, and necrotizing enterocolitis (NEC). The results underscore the significance of NETs in fighting infections and their association with complications like sepsis and delayed wound healing. The breakdown products of NETs as a diagnostic tool of the clinical course of acute appendicitis will also be discussed. Understanding NET formation in the pathophysiology can potentially help to find new therapeutic approaches such as the application of DNase and elastase inhibitors to change the clinical course of various diseases in pediatric surgery such as improvement of wound healing, adhesion formation, NEC, and many more.

Abstract Objective This article describes in vitro and in vivo studies that aimed to further characterize the biopharmaceutical properties and pharmacokinetic (PK) profile of vericiguat and to guide dosing recommendations. Methods Five open-label, phase I studies characterized the biopharmaceutical aspects of vericiguat, including absolute bioavailability, bioavailabilities of different formulations, dose proportionality, and food effect. Area under the curve (AUC) and maximum plasma concentrations (C max), determined by a noncompartmental analysis, were compared by analysis of variance, and a mixed-effects power model was used to assess dose proportionality. The effect of food on the dissolution of vericiguat was evaluated in vitro using media simulating the gastrointestinal tract under fed and fasted conditions. In vitro dissolution of intact vs crushed vericiguat tablet was assessed in quality control medium (HCl at pH 2), acetate buffer at pH 4.5, and phosphate buffer at pH 6.8. Results Dissolution of vericiguat increased under fed conditions. In healthy subjects, exposure (AUC and C max) increased ~ 40% with food vs fasted state (10 mg intact tablet) confirming a food effect on vericiguat bioavailability. Interindividual variability in exposure decreased ~ 20%, irrespective of meal type. Absolute bioavailability of vericiguat 10 mg (intact tablets, fed) was 93%. Vericiguat 2.5–10 mg demonstrated dose proportionality (intact tablets, fed) in healthy subjects. Dissolution studies showed no differences between the formulations, and this was confirmed with in vivo studies. Conclusion Vericiguat tablets should be administered with food and may be crushed for patients who have difficulty swallowing. Graphical Abstract

Abstract Background Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. Methods Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. Results Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. Conclusions Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Necrotizing enterocolitis (NEC) continues to be one of the most common causes of mortality and morbidity in preterm infants. Although not fully elucidated, studies suggest that prematurity, formula feeding, imbalanced vascular supply, and altered bacterial colonization play major roles in the pathogenesis of NEC. NEC is characterized by increased cytokine release and leukocyte infiltration. Recent data from preterm infants and animal models of NEC suggest that neutrophil extracellular traps (NETs) are released in intestinal tissue. The contribution of NETs in the pathogenesis and/or prevention/treatment of this disease continues to be controversial. Here, we review the available data on NETs release in NEC in human patients and in different NEC models, highlighting their potential contribution to pathology and resolution of inflammation. Here, we review the available data on NETs release in NEC in human patients and the different NEC models, highlighting their potential contribution to pathology or resolution of inflammation.

Abstract Vericiguat was developed for the treatment of symptomatic chronic heart failure (HF) in adult patients with reduced ejection fraction who are stabilized after a recent decompensation event. Guidelines recommend long‐acting nitrates, such as isosorbide mononitrate, for angina prophylaxis in chronic coronary syndromes (CCS), common comorbidities in HF. This study evaluated safety, tolerability, and the pharmacodynamic (PD) interaction between co‐administered vericiguat and isosorbide mononitrate in patients with CCS. In this phase Ib, double‐blind, multicenter study, patients were randomized 2:1 to receive vericiguat plus isosorbide mononitrate (n = 28) or placebo plus isosorbide mononitrate (n = 13). Isosorbide mononitrate was uptitrated to a stable dose of 60 mg once daily, followed by co‐administration with vericiguat (uptitrated every 2 weeks from 2.5 mg to 5 mg and 10 mg) or placebo. Thirty‐five patients completed treatment (vericiguat, n = 23; placebo, n = 12). Mean baseline‐ and placebo‐adjusted vital signs showed reductions of 1.4–5.1 mmHg (systolic blood pressure) and 0.4–2.9 mmHg (diastolic blood pressure) and increases of 0.0–1.8 beats per minute (heart rate) with vericiguat plus isosorbide mononitrate. No consistent vericiguat dose‐dependent PD effects were noted. The incidence of adverse events (AEs) was 92.3% and 66.7% in the vericiguat and placebo groups, respectively, and most were mild in intensity. Blood pressure and heart rate changes observed with vericiguat plus isosorbide mononitrate were not considered clinically relevant. This combination was generally well‐tolerated. Concomitant use of vericiguat with isosorbide mononitrate is unlikely to cause significant AEs beyond those known for isosorbide mononitrate.

Background: The purpose of this report is to describe the seminal case of a near-term human fetus with a life-threatening left diaphragmatic hernia that underwent fetoscopic tracheal occlusion (FETO) combined with fetoscopic partial removal of herniated bowel from the fetal chest by fetoscopic laparoschisis (FETO-LAP). Case summary: A life-threatening left diaphragmatic hernia (liver-up; o/e LHR of ≤25%; MRI lung volume ≤ 20%) was observed in a human fetus at 34 weeks of gestation. After counselling the mother about the high risks of postnatal demise if left untreated, the expected limitations of fetoscopic tracheal occlusion (FETO), and the previously untested option of combining FETO with fetoscopic laparoschisis, i.e., partial removal of the herniated bowel from the fetal chest (FETO-LAP), she consented to the latter novel treatment approach. FETO-LAP was performed at 36 + 5 weeks of gestation under general maternofetal anesthesia. Mother and fetus tolerated the procedure well. The neonate was delivered and the balloon removed on placental support at 37 + 2 weeks of gestation. On ECMO, a rapid increase in tidal volume was seen over the next eight days. Unfortunately, after this period, blood clots obstructed the ECMO circuit and the neonate passed away. Discussion: This seminal case shows that in a fetus with severe left diaphragmatic hernia, partial removal of the herniated organs from the fetal chest is not only possible by minimally invasive fetoscopic techniques but also well tolerated. As the effect of FETO alone is limited in saving severely affected fetuses, combining FETO with fetoscopic laparoschisis (FETO-LAP) offers a new therapeutic route with multiple, potentially life-saving implications.

(1) Background: Embryonal abdominal tumors are one of the most common entities of solid childhood cancer. The present study investigates the Health-Related Quality of Life (HRQoL) and the mental health of children to obtain a comprehensive picture of their health status and uncover a possible gap in healthcare. (2) Methods: The sample consisted of 54 children who were treated for embryonal abdominal tumors and a control group of 46 children who received uncomplicated outpatient surgery. The HRQoL and the mental health were assessed by the parent proxy reports of the questionnaires Pediatric Quality of Life Inventory (PedsQL) and Strengths and Difficulties Questionnaire (SDQ). (3) Results: Children with embryonal abdominal tumors showed significantly lower HRQoL and mental health values compared to the norm data. The index group showed lower values in the social subscales of HRQoL and mental health compared to the control group. (4) Conclusions: Embryonal abdominal tumors affect the well-being of children. There is still a gap in healthcare due to children’s HRQoL and mental health, especially regarding social development. It is essential to further advance the psychological care of children and improve their chances to develop social relationships.

IntroductionSevere burns cause unique pathophysiological alterations especially on the immune system. A murine scald model was optimized as a basis for the understanding of immunological reactions in response to heat induced injury. The understanding of the roles of neutrophil extracellular traps (NETs) and DNases will support the development of new surgical or pharmacological strategies for the therapy of severe burns.MethodsWe studied C57BL/6 mice (n=30) and employed four scalding protocols with varying exposure times to hot water. An additional scald group with a shorter observational time was generated to reduce mortality and study the very early phase of pathophysiology. At 24h or 72h, blood was drawn and tissue (wound, liver, lung, spleen) was analyzed for the presence of NETs, oxidative stress, apoptosis, bacterial translocation, and extracellular matrix re-organization. In addition, we analyzed the transcriptome from lung and liver tissues.ResultsExposure to hot water for 7s led to significant systemic and local effects and caused considerable late mortality. Therefore, we used an observation time of 24h in this groups. To study later phases of burns (72h) an exposure time of 6s is optimal. Both conditions led to significant disorganization of collagen, increased oxidative stress, NET formation (by immunodetection of H3cit, NE, MPO), apoptosis (cC3) and alterations of the levels of DNase1 and DNase1L3. Transcriptome analysis revealed remarkable alterations in genes involved in acute phase signaling, cell cohesion, extracellular matrix organization, and immune response.ConclusionWe identified two scald models that allow the analysis of early (24h) or late (72h) severe burn effects, thereby generating reproducible and standardized scald injuries. The study elucidated the important involvement of neutrophil activity and the role of NETs in burns. Extensive transcriptome analysis characterized the acute phase and tissue remodeling pathways involved in the process of healing and may serve as crucial basis for future in-depth studies.

Besides performing phagocytosis and degranulation, neutrophils are capable of eliminating microorganisms by releasing neutrophil extracellular traps (NETs). NET formation was found to be associated with increased mortality in sepsis. During sepsis levels of interleukin 1 (IL-1), a cytokine, increases significantly and also was associated with increased mortality. Blocking of the interleukin 1 (IL-1) receptor by anakinra leads to less NET formation in gout patients. However, NET formation is crucial during infection by trapping pathogens and thereby slowing the process. Total or early blocking of cascades leading to NETs may lead to aggravation of infection in otherwise mild cases. The dose- and time-dependent effect of the IL-1 receptor antagonist anakinra was tested on spontaneous, lipopolysaccharide (LPS)-induced and phorbol-12-myristate 13-acetate (PMA)-induced formation of NETs in vitro . Quantitative detection of NETs was performed for NETspecific proteins and cell-free DNA. Immunostained microscopy imaging was used for visualization. Our study shows a dose- and time-dependent inhibitory effect of anakinra that involves the change of intracellular calcium mobilization on the formation of NETs in vitro for PMA-stimulated neutrophils but not for LPS-stimulated neutrophils. It may be useful for treatment of sepsis as part of a multimodal treatment concept, but it seems that timing and dose need to be carefully chosen.

Abstract Background COVID-19 has affected our society at large, particularly vulnerable groups, such as children suffering from rare diseases and their parents. However, the psychosocial influences of COVID-19 on these have yet to be investigated. As such, the study’s goal was to evaluate the health-related quality of life (HRQoL), quality of life (QoL), and mental health of children with rare congenital surgical diseases and their parents during the COVID-19 pandemic and lockdown measures. Methods A survey of n = 210 parents of children with rare congenital surgical diseases and a control group of n = 88 parents of children without rare diseases was conducted cross-sectionally between April 2020 to April 2021. Data on HRQoL, QoL, and mental health was collected using standardized psychometric questionnaires for children and parents presenting to the pediatric surgery department at a university hospital. Results Mothers of children with rare pediatric surgical diseases showed significantly lower QoL and significantly higher impairment in mental health than a control group and norm data. For fathers, this was solely the case for their QoL. Children’s parent-reported HRQoL and mental health were partially impaired. Social and disease-specific risk factors of the respective outcomes in affected families were identified through regression analysis models. Conclusion Parents of children with rare diseases report severe psychosocial impairment regarding themselves and their children during the COVID-19 pandemic. Therefore, affected families should receive attention and supportive care in the form of a family-center approach to alleviate the additional burden of the COVID-19 pandemic.

Abstract Background Previous research has supported the utility of the Double ABCX model of family adaptation for parents in various diseases. Nonetheless, it remains unclear how raising a child with rare congenital surgical diseases impacts the mental health of both mothers and fathers. Methods The potential predictors of maternal and paternal mental health in a German sample of 210 parents of children with rare congenital surgical diseases were investigated. Parents were investigated cross-sectionally utilizing standardized psychometric questionnaires that assessed factors attributed to parental adaptation within the Double ABCX model. Results Stressor pile-up, family functioning, perceived stress, and mental health were positively associated with mothers and fathers. However, further analyses revealed that family functioning, social support, and perceived stress fully mediated the positive association between stressor pile-up and mental health in mothers, but not fathers. Conclusion Our findings suggest that parental adaptation to a rare congenital surgical disease in their children may be improved by increased intra- and extrafamilial resources and decreased perceived family-related stress in mothers, but not fathers. Our results may help to identify gender-specific factors that may guide clinicians and future interventions.

Introduction Childhood cancer is a leading cause of death. It is unclear whether the COVID-19 pandemic has impacted childhood cancer mortality. In this study, we aimed to establish all-cause mortality rates for childhood cancers during the COVID-19 pandemic and determine the factors associated with mortality.Methods Prospective cohort study in 109 institutions in 41 countries. Inclusion criteria: children

(1) Background: As increases in intra-abdominal pressure (IAP) result in irreversible tissue damage, monitoring IAP in critically ill patients using the common urinary bladder catheter method is essential. However, this method can result in complications and is not suitable for very low birth weight neonates. The aim of this study was to establish a non-invasive and accurate method to detect IAP changes using an animal model. (2) Methods: IAP changes via intra-abdominal air application (up to 20 mmHg) were measured in 19 Wistar rats via an intra-abdominally placed intracranial pressure probe. Concurrently, abdominal surface tension was measured using a Graseby capsule (GC). (3) Results: A high correlation between abdominal wall distension and IAP (r = 0.9264, CI 0.9249–0.9279) was found for all subjects. (4) Conclusions: IAP changes in rats can be detected non-invasively using a GC. However, further studies are necessary to assess whether IAP changes can be measured using a GC in the neonatal population.

Abstract Background X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation. Results To identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression. Conclusions Here, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.

ObjectiveIn patients with a congenital diaphragmatic hernia (CDH), postoperative small bowel obstruction (SBO) is a life-threatening event. Literature reports an incidence of SBO of 20% and an association with patch repair and ECMO treatment. Adhesions develop due to peritoneal damage and underly various biochemical and cellular processes. This longitudinal cohort study is aimed at identifying the incidence of SBO and the risk factors of surgical, pre-, and postoperative treatment.MethodsWe evaluated all consecutive CDH survivors born between January 2009 and December 2017 participating in our prospective long-term follow-up program with a standardized protocol.ResultsA total of 337 patients were included, with a median follow-up of 4 years. SBO with various underlying causes was observed in 38 patients (11.3%) and significantly more often after open surgery (OS). The majority of SBOs required surgical intervention (92%). Adhesive SBO (ASBO) was detected as the leading cause in 17 of 28 patients, in whom surgical reports were available. Duration of chest tube insertion [odds ratio (OR) 1.22; 95% CI 1.01–1.46, p = 0.04] was identified as an independent predictor for ASBO in multivariate analysis. Beyond the cut-off value of 16 days, the incidence of serous effusion and chylothorax was higher in patients with ASBO (ASBO/non-SBO: 2/10 vs. 3/139 serous effusion, p = 0.04; 2/10 vs. 13/139 chylothorax, p = 0.27). Type of diaphragmatic reconstruction, abdominal wall closure, or ECMO treatment showed no significant association with ASBO. A protective effect of one or more re-operations has been detected (RR 0.16; 95% CI 0.02–1.17; p = 0.049).ConclusionThoracoscopic CDH repair significantly lowers the risk of SBO; however, not every patient is suitable for this approach. GoreTex®-patches do not seem to affect the development of ASBO, while median laparotomy might be more favorable than a subcostal incision. Neonates produce more proinflammatory cytokines and have a reduced anti-inflammatory capacity, which may contribute to the higher incidence of ASBO in patients with a longer duration of chest tube insertion, serous effusion, chylothorax, and to the protective effect of re-operations. In the future, novel therapeutic strategies based on a better understanding of the biochemical and cellular processes involved in the pathophysiology of adhesion formation might contribute to a reduction of peritoneal adhesions and their associated morbidity and mortality.

Objectives Paediatric cancer is a leading cause of death for children. Children in low-income and middle-income countries (LMICs) were four times more likely to die than children in high-income countries (HICs). This study aimed to test the hypothesis that the COVID-19 pandemic had affected the delivery of healthcare services worldwide, and exacerbated the disparity in paediatric cancer outcomes between LMICs and HICs.Design A multicentre, international, collaborative cohort study.Setting 91 hospitals and cancer centres in 39 countries providing cancer treatment to paediatric patients between March and December 2020.Participants Patients were included if they were under the age of 18 years, and newly diagnosed with or undergoing active cancer treatment for Acute lymphoblastic leukaemia, non-Hodgkin’s lymphoma, Hodgkin lymphoma, Wilms’ tumour, sarcoma, retinoblastoma, gliomas, medulloblastomas or neuroblastomas, in keeping with the WHO Global Initiative for Childhood Cancer.Main outcome measure All-cause mortality at 30 days and 90 days.Results 1660 patients were recruited. 219 children had changes to their treatment due to the pandemic. Patients in LMICs were primarily affected (n=182/219, 83.1%). Relative to patients with paediatric cancer in HICs, patients with paediatric cancer in LMICs had 12.1 (95% CI 2.93 to 50.3) and 7.9 (95% CI 3.2 to 19.7) times the odds of death at 30 days and 90 days, respectively, after presentation during the COVID-19 pandemic (p

Abstract Lithocholic bile acid (LCA) has been reported to selectively kill cancer cells within many tumor cell lines including neuroblastoma or glioblastoma. Wilms’ tumor shares similarities with neuro- and glioblastoma. Hence, the aim of the study was to evaluate the effects of LCA on nephroblastoma. To test the effects of LCA, nephroblastoma cell line WT CLS1 was used. SK NEP1 was tested as well. It was originally classified as a nephroblastoma cell line but was meanwhile reclassified as an ewing sarcoma cell line. As control cell lines HEK 293 from embryonic kidney and RC 124 from adult kidney tissue as well as podocytes were used. The effects were evaluated using proliferation assay, caspase activity assay, FACS and Western blot. LCA showed a dose and time-dependent selective effect inducing apoptosis in nephroblastoma cells. However, these effects were not limited to the nephroblastoma cell line but also affected control kidney cell lines and the sarcoma cells; only podocytes are significantly less affected by LCA (at dosages

Abstract Appendicitis is one of the most frequent emergencies in pediatric surgery, yet current biomarkers for diagnosis are unspecific and have low predictive values. As neutrophils and extracellular traps (ETs) are an essential component of the immune defense against bacterial infections, and appendicitis is considered an inflammation reaction of the appendix, we hypothesized that neutrophil activation and NET formation play an essential role in appendicitis development and maintenance. Therefore, this pilot study aimed to establish a murine model of appendicitis and to evaluate ETs markers to diagnose appendicitis in mice and humans. The study used 20 (12 appendicitis- and 8 controls) 6-week old mice which underwent advanced appendicitis induction using a modified caecal ligation puncture procedure. During the study, cell-free DNA, neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated Histone H3 (H3cit) were assessed. Additionally, samples of 5 children with histologically confirmed appendicitis and 5 matched controls with catarrhal appendicitis, were examined for the same biomarkers. Moreover, NE, MPO, and H3cit were assessed histologically via immunofluorescence in mice and humans. All mice in the appendicitis group developed an advanced form of appendicitis with focal peritonitis. In mice and humans with appendicitis, markers of neutrophil activation and ETs formation (especially cfDNA, NE and H3cit) were significantly elevated in blood and tissue compared to controls. Ultimately, biomarkers correlated extremely well with tissue expression and thus disease severity. It appears that neutrophil activation and possibly NETs contribute to appendicitis development and biomarkers of neutrophil activation and ET formation reflect disease severity and thus could be used as biomarkers for appendicitis. However, large prospective clinical studies are needed to confirm our findings.

BackgroundNeutrophil extracellular traps (NETs)—as double-edged swords of innate immunity—are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs.MethodsWe analysed the prolongation of neutrophil survival in vitro under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO).ResultsUntreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α.ConclusionsG-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions.

IntroductionEsophageal stenoses are frequent complications after esophageal atresia surgery as well as after acid, alkali and battery ingestion. Worldwide, repeated balloon dilatations are the most frequently performed procedures for these stenoses. In most cases the stenoses can be dilated sufficiently to allow adequate enteral nutrition. Until recently, age dependent esophageal lumen size has not been established; which was aim of the current study.MethodsAll children in whom an esophageal contrast imaging was performed between 1/2011 and 5/2021 were included. The width was measured by two investigators at two measuring points in two planes, the diameter was calculated and plotted against the respective weight of the child. Bland-Altmann plots have confirmed the validity of the measurements of both investigators.ResultsEsophagus diameter was measured in more than 100 children. The resulting curves show a very good correlation with weight (upper measuring point: r = 0.86743, p < 0.0001; lower measuring point: r = 0.80593, p < 0.0001).ConclusionThese results are the first to define the esophageal diameter in children. The results of this study may guide physicians performing esophageal interventions including dilatations in future.

Background: Severe infections that culminate in sepsis are associated with high morbidity and mortality. Despite continuous efforts in basis science and clinical research, evidence based-therapy is mostly limited to basic causal and supportive measures. Adjuvant therapies often remain without clear evidence. The objective of this study was to evaluate the septic volvulus ischemia-reperfusion model in comparison to two already established models and the role of neutrophil extacellular traps (NETs) in this model. Methods: The technique of the murine model of midgut volvulus was optimized and was compared to two established models of murine sepsis, namely cecal ligation and puncture (CLP) and intra-peritoneal (i.p.) injection of lipopolysaccharide (LPS). Results: Midgut volvulus for 15 min caused a comparable mortality (38%) as CLP (55%) and peritoneal LPS injection (25%) at 48 h. While oxidative stress was comparable, levels of circulating free DNA (cfDNA), and splenic/hepatic and pulmonary translocation of bacteria were decreased and increased, respectively at 48 h. DNases were increased compared to the established models. Proteomic analysis revealed an upregulation of systemic Epo, IL-1b, Prdx5, Parp1, Ccl2 and IL-6 at 48 h in comparison to the healthy controls. Discussion and Conclusion: Midgut volvulus is a stable and physiological model for sepsis. Depending on the duration and subsequent tissue damage, it represents a combination of ischemia-reperfusion injury and hyperinflammation.

Abstract Background While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells. Results Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA. Conclusion Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.

Since there are no data available on the influence of the time point of ECMO initiation on morbidity and mortality in patients with congenital diaphragmatic hernia (CDH), we investigated whether early initiation of ECMO after birth is associated with a beneficial outcome in severe forms of CDH. All neonates with CDH admitted to our institution between 2010 until 2020 and undergoing ECMO treatment were included in this study and divided into four different groups: (1) ECMO initiation < 12 h after birth (n = 143), (2) ECMO initiation between 12–24 h after birth (n = 31), (3) ECMO initiation between 24–120 h after birth (n = 48) and (4) ECMO initiation > 120 h after birth (n = 14). The mortality rate in the first (34%) and fourth group (43%) was high and in the second group (23%) and third group (12%) rather low. The morbidity, characterized by chronic lung disease (CLD), did not differ significantly in the three groups; only patients in which ECMO was initiated >120 h after birth had an increased rate of severe CLD. Our data, although not randomized and limited due to small study groups, suggest that very early need for ECMO and ECMO initiation > 120 h after birth is associated with increased mortality.

One potential comorbidity after congenital diaphragmatic hernia (CDH) is gastroesophageal reflux (GER), which can have a substantial effect on patients’ quality of life, thriving, and complications later in life. Efforts have been made to reduce gastroesophageal reflux with a preventive anti-reflux procedure at the time of CDH repair. In this follow-up study of neonates participating in a primary RCT study on preventive anti-reflux surgery, symptoms of GER were assessed longitudinally. Long-term data with a median follow-up time of ten years was available in 66 patients. Thirty-one neonates received an initial fundoplication. Secondary anti-reflux surgery was necessary in 18% and only in patients with large defects. It was required significantly more often in patients with intrathoracic herniation of liver (p = 0.015) and stomach (p = 0.019) and patch repair (p = 0.03). Liver herniation was the only independent risk factor identified in multivariate regression analysis. Primary fundopexy and hemifundoplication did not reveal a protective effect regarding the occurrence of GER symptoms, the need for secondary antireflux surgery or the gain of body weight regardless of defect size neither in the short nor in the long term. Symptoms of GER must be assessed carefully especially in children with large defects, as these are prone to require secondary anti-reflux surgery in the long-term. Routine evaluation of GER including endoscopy and impedance measurement should be recommended especially for high-risk patients.

Background: Pilonidal sinus (PS) disease frequently occurs in adolescents and young adults, and in many cases involves wide excision or local flaps as treatment. These treatments are associated with a significant recurrence rate, a long healing time, and thus absence from school or work. The hybrid technique, which is a combination of side-swing plasty with negative-pressure wound therapy (NPWT) may improve these outcomes. The aim of the study was to compare the latter with other current methods.Methods: Children presenting with a pilonidal sinus to two referral centers for pediatric surgery from January 2017 till June 2019 and subsequent (1) slide-swing plasty, (2) open excision, or (3) slide-swing plasty in combination with NPWT were included in this retrospective study. Type of therapy, number of interventions, duration of hospitalization, complications, and recurrence rate were recorded. In addition, data was retrieved from the national diagnosis-related group for inpatient statistics, for all patients who underwent surgery for pilonidal sinus in 2015 and 2016.Results: In total, 85 children were included, with a mean age of 15 years and a near equal gender distribution (53% female). The minimum follow-up was 1 year. In 56% open resection was performed, while 18% underwent a slide-swing plasty and 26% a slide-swing plasty in combination with NPWT. While the hybrid technique was superior regarding recurrence rate in comparison to open excision (24 vs. 5%, p = 0.047), it had significantly longer hospital stay [17.41 (15.63) vs. 3.65 (1.68) days, p < 0.001] and number of interventions [4.14 (4.07) vs. 1.04 (0.29), p < 0.001].Conclusions: Management of PS disease using slide-swing plasty in combination with NPWT is an effective treatment and is associated with low recurrence rate and minimal morbidity. However, this type of treatment is accompanied by an elongated hospitalization time and more frequent interventions. A diligent case by case evaluation and thorough patient counseling is thus necessary when choosing the right technique for the treatment of PS disease.

BackgroundNeutrophils are the first responders in wound healing after injury that mediate pro- and anti-inflammatory activities i.a. through the formation of extracellular traps (NETs). However, excessive NETs presence in wound tissue can cause local hyperinflammation and -coagulation resulting in delayed wound healing. To improve wound healing, we aimed to examine the role of NETs and DNase1 on primary and secondary wound healing.MethodsThe study included 93 C57BL/6 mice, with 3 different genotypes: wildtype, Pad4-, and DNase1-Knockout (KO). Pad4-KO mice show limited NETs formation, while DNase1-KO mice cannot disintegrate them. All 3 genotypes were included in (1) a laparotomy group and (2) a thermal injury group. Animals in both groups either received DNase1 or a vehicle i.p. post wound induction and wound assessment and euthanasia were conducted. Laparotomy and burn scars were assessed using the stony brook scar evaluation scale and modified Yeong scale respectively. Tissue was analyzed histologically using H&E staining. Ly6g, Collagen I and III, SMA, and Fibrinogen were visualized and neutrophils activation (NE, MPO) and NETs (H3cit) formation assessed.ResultsAll animals survived with no complications. DNase1 treatment led to a significantly improved scar appearance in both groups, which was also seen in Pad4-KO mice. In the laparotomy group DNase1 improved collagen deposition and fibrin concentration was significantly reduced by DNase1 treatment. Markers of neutrophil activation were significantly reduced in the treatment and Pad4-KO group. In the thermal injury group wound closure time was significantly reduced after DNase1 treatment and in the Pad4-KO group. Even though inflammation remained high in the thermal injury model over time, neutrophil activation and NETs formation were significantly reduced by DNase1 treatment compared to controls.DiscussionPrimary and secondary intention wound healing is improved by targeting NETs through DNase1 treatment or genetic KO, as assessed by wound closure time and scar appearances. Additionally, wound stability was not affected by DNASE treatment. The results suggest that overall wound healing is accelerated and DNase1 appears to be a promising option to reduce scar formation; which should be evaluated in humans.

Background: Necrotizing enterocolitis (NEC) is an often-fatal neonatal disease involving intestinal hyperinflammation leading to necrosis. Despite ongoing research, (1) conflicting results and (2) comorbidities of NEC patients make early NEC detection challenging and may complicate therapy development. Most research suggests that NEC pathogenesis is multifactorial, involving a combination of (1) gut prematurity; (2) abnormal bacterial colonization; and (3) ischemia-reperfusion (I/R) injury. As neutrophil extracellular traps (NETs) partially mediate I/R injury and drive inflammation in NEC, we hypothesized that NETs contribute to NEC development; particularly in cardiac patients.Methods: A retrospective analysis of baseline characteristics, clinical signs, laboratory parameters, and imaging was conducted for surgically verified NEC cases over 10 years. Patients were stratified into two groups: (1) prior medically or surgically treated cardiac disease (cardiac NEC) and (2) no cardiac comorbidities (inflammatory NEC). Additionally, histology was reassessed for neutrophil activation and NETs formation.Results: A total of 110 patients (cNEC 43/110 vs. iNEC 67/110) were included in the study, with cNEC neonates being significantly older than iNEC neonates (p = 0.005). While no significant differences were found regarding clinical signs and imaging, laboratory parameters revealed that cNEC patients have significantly increased leucocyte (p = 0.024) and neutrophil (p < 0.001) counts. Both groups also differed in pH value (p = 0.011). Regarding histology: a non-significant increase in staining of myeloperoxidase within the cNEC group could be found in comparison to iNEC samples. Neutrophil elastase (p = 0.012) and citrullinated histone H3 stained (p = 0.041) slides showed a significant markup for neonates diagnosed with cNEC in comparison to neonates with iNEC.Conclusion: The study shows that many standardized methods for diagnosing NEC are rather unspecific. However, differing leucocyte and neutrophil concentrations for iNEC and cNEC may indicate a different pathogenesis and may aid in diagnosis. As we propose that iNEC is grounded rather in sepsis and neutropenia, while cNEC primarily involves I/R injuries, which involves neutrophilia and NETs formation, it is plausible that I/R injury due to interventions for cardiac comorbidities results in pronounced neutrophil activation followed by a hyperinflammation reaction and NEC. However, prospective studies are necessary to validate these findings and to determine the accuracy of the potential diagnostic parameters.

Background: It is not only important for counseling purposes and for healthcare management. This study investigates the prediction accuracy of an artificial intelligence (AI)-based approach and a linear model. The heuristic expecting 1 day of stay per percentage of total body surface area (TBSA) serves as the performance benchmark.Methods: The study is based on pediatric burn patient's data sets from an international burn registry (N = 8,542). Mean absolute error and standard error are calculated for each prediction model (rule of thumb, linear regression, and random forest). Factors contributing to a prolonged stay and the relationship between TBSA and the residual error are analyzed.Results: The random forest-based approach and the linear model are statistically superior to the rule of thumb (p < 0.001, resp. p = 0.009). The residual error rises as TBSA increases for all methods. Factors associated with a prolonged LOS are particularly TBSA, depth of burn, and inhalation trauma.Conclusion: Applying AI-based algorithms to data from large international registries constitutes a promising tool for the purpose of prediction in medicine in the future; however, certain prerequisites concerning the underlying data sets and certain shortcomings must be considered.

BackgroundNeutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of decondensed chromatin and about 30 enzymes and peptides. Some components, such as neutrophil elastase (NE) and myeloperoxidase (MPO), present antimicrobial but also cytotoxic properties, leading to tissue injury. Many inflammatory diseases are associated with NETs, and their final role has not been identified. Pulmonary surfactant is known to have immunoregulatory abilities that alter the function of adaptive and innate immune cells. The aim of this study was to investigate the hypothesis that natural surfactant preparations inhibit the formation of NETs.MethodsThe effect of two natural surfactants (Alveofact® and Curosurf®) on spontaneous and phorbol-12-myristate-13-acetate–induced NET formation by neutrophils isolated by magnetic cell sorting from healthy individuals was examined. NETs were quantitatively detected by absorption and fluorometric-based assays for the NET-specific proteins (NE, MPO) and cell-free DNA. Immunofluorescence microscopy images were used for visualization.ResultsBoth surfactant preparations exerted a dose-dependent inhibitory effect on NET formation. Samples treated with higher concentrations and with 30 min pre-incubation prior to stimulation with phorbol-12-myristate-13-acetate had significantly lower levels of NET-specific proteins and cell-free DNA compared to untreated samples. Immunofluorescence microscopy confirmed these findings.ConclusionsThe described dose-dependent modulation of NET formation ex vivo suggests an interaction between exogenous surfactant supplementation and neutrophil granulocytes. The immunoregulatory effects of surfactant preparations should be considered for further examination of inflammatory diseases.

Introduction: Testicular torsion (TT) is a common emergency that warrants immediate exploration to prevent infertility or testicular loss. To improve diagnostic reliability, various scoring systems have been published. The aim of this study was to evaluate and validate different testicular torsion scores in a large cohort of children with acute scrotum.Methods: Retrospective analysis of all male children that were admitted for acute scrotum at the Pediatric Surgery Department of the Altonaer Kinderkrankenhaus and University medical Center Hamburg-Eppendorf from 01/2013 to 03/2019. Two testicular torsion scores (Boettcher Alert Score, Testicular Workup for Ischemia and Suspected Torsion Score) were applied to all data sets. Furthermore, an artificial intelligence (AI)-based score was developed and compared to the two current scores.Results: In total, 460 boys were included in the study. Of those, 48 (10.4%) had TT. Children with TT suffered most often from short duration of pain, nausea and vomiting, high riding testicle and absent cremasteric reflex. The BALS and the AI-based score had excellent predictive values and all patients with TT would have been detected.Conclusion: The BAL and the AI score show excellent predictive capabilities and may be used to identify all cases of TT in a pediatric population. The scores are easy to apply. As the BALS was slightly better, we advocate to use this score but to validate our findings in prospective multicenter studies.

The aim of this paper is to assess the effectiveness and perioperative complications of splenic surgeries in children. In 41 splenectomies, an anterior abdominal laparoscopic approach was used, with 35 including a partial laparoscopic splenectomy. Of these, three needed a conversion to open. Six patients had a total splenectomy, three of which were open. Patients ranged in age from 5 to 18 years. Splenectomy was performed for a variety of causes, including hereditary spherocytosis (n = 20), splenic cysts (n = 13), sickle cell disease (n = 3), primary malignancy (n = 1), sepsis (n = 1), embolism (n = 1), anemia (n = 1), and hypersplenism (n = 1). The average length of stay was 7.6 days, and the average operation time was 169.3 min. Pleural effusion in the left hemithorax was found in 31.6% of the patients, with 5.3% requiring a thorax drain. The majority of patients had the highest platelet count two weeks after surgery. There was no evidence of wound infection, pancreatic leak, colon perforation, or postoperative sepsis. The most encountered perioperative complication was bleeding with the need of transfusion (n = 6), and one patient needed a diaphragm repair. A partial splenectomy (PS) can be a difficult procedure with a steep learning curve. For most children who require a splenic operation, this should be the primary procedure of choice.

Background: Acute appendicitis represents the most frequent reason for abdominal surgery in children. Since diagnosis can be challenging various scoring systems have been published. The aim of this study was to evaluate and validate (and improve) different appendicitis scores in a very large cohort of children with abdominal pain.Methods: Retrospective analysis of all children that have been hospitalized due to suspected appendicitis at the Pediatric Surgery Department of the Altonaer Children's Hospital and University Medical Center Hamburg-Eppendorf from 01/2018 until 11/2019. Four different appendicitis scores (Heidelberg Appendicitis Score, Alvarado Score, Pediatric Appendicitis Score and Tzanakis Score) were applied to all data sets. Furthermore, the best score was improved and artificial intelligence (AI) was applied and compare the current scores.Results: In 23 months, 463 patients were included in the study. Of those 348 (75.2%) were operated for suspected appendicitis and in 336 (96.6%) patients the diagnosis was confirmed histopathologically. The best predictors of appendicitis (simple and perforated) were rebound tenderness, cough/hopping tenderness, ultrasound, and laboratory results. After modifying the HAS, it provided excellent results for simple (PPV 95.0%, NPV 70.0%) and very good for perforated appendicitis (PPV 34.4%, NPV 93.8%), outperforming all other appendicitis score.Discussion: The modified HAS and the AI score show excellent predictive capabilities and may be used to identify most cases of appendicitis and more important to rule out perforated appendicitis. The new scores outperform all other scores and are simple to apply. The modified HAS comprises five features that can all be assessed in the emergency department as opposed to current scores that are relatively complex to utilize in a clinical setting as they include of up to eight features with various weighting factors. In conclusion, the modified HAS and the AI score may be used to identify children with appendicitis, yet prospective studies to validate our findings in a large mutli-center cohorts are needed.

Background: Infants with ileostomies often suffer from sodium depletion, ultimately leading to a failure to thrive. Moreover, early-infantile microbial dysbiosis may potentially aggravate weight faltering. Given that sodium supplementation has been used to restore weight gain and feeding practices largely determine infantile microbiota, the current study investigated the effect of sodium chloride (NaCl) on weight gain and intestinal microbiome in infants with jejuno- and ileostomies.Methods: A prospective cohort study including 24 neonates with enterostomies compared 19 subjects receiving oral NaCl (5.85%) to five subjects without supplementation with respect to postoperative changes in thrive and the intestinal microbiome.Results: Infants receiving NaCl after enterostomy-surgery showed vastly improved weight gain and an increased abundance of Lactobacillus in fecal samples, as compared to subjects without oral supplement who displayed decreasing percentiles for weight and did not reveal a higher abundance of probiotic strains within the ostomy effluent. Contrarily, Klebsiella was equally enriched in supplemented infants, reflecting a higher susceptibility for infections in preterm neonates.Discussion: Our findings support oral NaCl supplementation as a mainstay of postoperative treatment in infants with small bowel ostomies who are predisposed to suffer from a sodium depletion-associated failure to thrive. Not only does NaCl promote weight gain by increasing glucose resorption, but it also appears to induce microbial restoration by enhancing the abundance of health-promoting probiotic bacteria. This finding has an even greater significance when facing an elevated Klebsiella/Bifidobacteria (K/B) ratio, believed to represent an early-life microbial biomarker for development of allergic disease.

Introduction: Early-onset sepsis in neonates potentially results in substantial morbidity and mortality. A key player in sepsis a neutrophil extracellular traps (NETs) to limit dissemination of pathogens. Aim of this study was to evaluate markers of NET formation in umbilical cord blood as a predictor of neonatal sepsis.Methods: Prospective study including term and preterm neonates. Umbilical cord blood samples were obtained immediately after birth and following markers of inflammation and NET formation were assessed: complete blood count, C-reactive protein (CRP), interleukin 6 (IL-6), levels of cell-free DNA (cfDNA), neutrophil elastase (NE), and myeloperoxidase (MPO). The study population included neonates with confirmed early-onset sepsis and propensity score matched controls.Results: Umbilical cord blood samples of 491 neonates were obtained, of whom 17 neonates (n = 17) presented clinical and laboratory signs of infection within the first 72 h postpartum. Seventeen neonates without infection were matched as controls. IL-6 differed significantly between both groups, whereas other infection parameters such as CRP and neutrophil levels, and in particular the NET surrogate markers (cfDNA, NE, MPO), did not show any significant differences.Conclusion: NET markers in umbilical cord blood appear to not predict the onset of neonatal sepsis. These findings probably result from the neonates‘ inability or delayed ability to form NETs, which is suspected to be a main reason for the increased risk of severe infections in neonates, but is also assumed to prevent negative NET-mediated consequences during perinatal adaptation.

Introduction: An early and accurate diagnosis of early onset neonatal sepsis (EONS) and late onset neonatal sepsis (LONS) is essential to improve the outcome of this devastating conditions. Especially, preterm infants are at risk. Reliable biomarkers are rare, clinical decision-making depends on clinical appearance and multiple laboratory findings. Markers of NET formation and NET turnover might improve diagnostic precision. Aim of this study was to evaluate the diagnostic value of NETs in sepsis diagnosis in neonatal preterm infants. Methods: Plasma samples of neonatal preterm infants with suspected sepsis were collected. Blood samples were assayed for markers of NET formation and NET turnover: cfDNA, DNase1, nucleosome, NE, and H3Cit. All clinical findings, values of laboratory markers, and epidemiological characteristics were collected retrospectively. Two subpopulations were created to divide EONS from LONS. EMA sepsis criteria for neonatal sepsis were used to generate a sepsis group (EMA positive) and a control group (EMA negative). Results: A total of 31 preterm neonates with suspected sepsis were included. Out of these, nine patients met the criteria for sepsis according to EMA. Regarding early onset neonatal sepsis (3 EONS vs. 10 controls), cfDNA, DNase I, nucleosome, and CRP were elevated significantly. H3Cit and NE did not show any significant elevations. In the late onset sepsis collective (6 LONS vs. 12 controls), cfDNA, DNase I, and CRP differed significantly compared to control group.

Staphylococcal bi-component pore-forming toxins, also known as leukocidins, target and lyse human phagocytes in a receptor-dependent manner. S-components of the leukocidins Panton-Valentine leukocidin (PVL), γ-haemolysin AB (HlgAB) and CB (HlgCB), and leukocidin ED (LukED) specifically employ receptors that belong to the class of G-protein coupled receptors (GPCRs). Although these receptors share a common structural architecture, little is known about the conserved characteristics of the interaction between leukocidins and GPCRs. In this study, we investigated host cellular pathways contributing to susceptibility towards S. aureus leukocidin cytotoxicity. We performed a genome-wide CRISPR/Cas9 library screen for toxin-resistance in U937 cells sensitized to leukocidins by ectopic expression of different GPCRs. Our screen identifies post-translational modification (PTM) pathways involved in the sulfation and sialylation of the leukocidin-receptors. Subsequent validation experiments show differences in the impact of PTM moieties on leukocidin toxicity, highlighting an additional layer of refinement and divergence in the staphylococcal host-pathogen interface. Leukocidin receptors may serve as targets for anti-staphylococcal interventions and understanding toxin-receptor interactions will facilitate the development of innovative therapeutics. Variations in the genes encoding PTM pathways could provide insight into observed differences in susceptibility of humans to infections with S. aureus.

Biomarkers are increasingly used to provide decision making data early in phase I by showing Proof of Mechanism or Proof of Concept (PoM/PoC). For antihypertensive agents, the administration of multiple doses (md) to hypertensive patients is assumed to be necessary for an early go/no-go decision. We compared the effects of an Angiotensin II receptor antagonist (ARA) on Plasma Renin and blood pressure (BP) following an oral single dose (sd) and once daily md for seven days to healthy volunteers and patients with essential hypertension (diastolic BP 95 mmHg to 114 mmHg; systolic BP 130 mmHg to 200 mmHg). Methods: 5–12 healthy male subjects/dose received 10 mg to 300 mg ARA sd and 50 to 300 mg md for 7 days; patients (9–10/dose) received 20 mg–400 mg ARA for 7 days. The studies were designed as randomized, singleblind, placebo-controlled, group comparison or crossover dose-escalation studies. Plasma Renin and BP were monitored up to 24 hours after dosing. Results: Plasma Renin showed a high interindividual variability in both healthy volunteers and patients. Healthy subjects showed a dose- and time-related increase in plasma Renin after sd from 40 mg to 300 mg and md of 50 mg to 300 mg (p < 0.05 for doses of 200 mg and 300 mg). In patients, increases in plasma Renin occurred at 8 hours and beyond starting at sd of 100 mg and md of 50 mg (p < 0.05 for the dose of 400 mg). While healthy volunteers showed no relevant decrease in BP, in hypertensive patients a reduction in BP in doses of 100 mg to 400 mg occurred (p < 0.05); effects were more pronounced after md compared to sd. Conclusion: Early PoM for an antihypertensive agent can be shown by use of laboratory biomarkers following sd to healthy subjects. PoC can be achieved after sd in hypertensive patients. Administration of sd to healthy volunteers is sufficient for an early go/no-decision.

Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα(-), PR(-), and Her2(-), named accordingly "Triple-Negative" (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5-12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygro(R)) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50-100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships.

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.