The Dual Role of Neutrophil Extracellular Traps (NETs) in Sepsis and Ischemia-Reperfusion Injury: Comparative Analysis across Murine Models.

A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for peptidyl arginine deiminase-4 (PAD4-KO) or DNase1 and 1L3 (DNase1/1L3-DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in PAD4-KO and DNase1/1L3-DKO mice, when compared to WT mice, respectively. PAD4-KO mice showed decreased formation of NETs and ROS, while DNase1/1L3-DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus).

NETworking for Health and in Disease: Neutrophil Extracellular Traps in Pediatric Surgical Care.

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.

Pleural Effusion and Chylothorax in Congenital Diaphragmatic Hernia-Risk Factors, Management and Outcome.

Background: Pleural effusion and chylothorax are common complications in the treatment of congenital diaphragmatic hernia (CDH). We set out to identify risk factors for chylothorax development in patients with CDH and to investigate the association of pleural effusion and chylothorax with neonatal morbidity and mortality. Methods: In this retrospective cohort study, we included 396 neonates with CDH treated at our institution between January 2013 and June 2019. Preoperative and postoperative chest radiographs and clinical data were evaluated and correlated with morbidity, complications and mortality. Results: Laboratory-confirmed chylothorax occurred in 58 (18.6%) of all CDH cases. Pleural effusion was frequently observed as a postoperative complication but also occurred as a pre-existing condition. Neonates with large defects of size C and D, patch repair, the need for presurgical and/or postsurgical ECMO support, pulmonary hypertension, liver-up phenomenon and lower relative fetal lung volume were associated with higher occurrences of chylothorax. After stepwise logistic regression, larger CDH defects (p < 0.0001) and the need for postsurgical ECMO (p = 0.0158) remained significant risk factors for CTX to occur (AUC 0.71). The same potential risk factors were used to assess their association with both presurgical and postsurgical pleural effusion. After stepwise logistic regression, only the need for presurgical ECMO remained significantly associated with presurgical PE (p < 0.01, AUC 0.65) and patch repair as the therapeutic intervention remained significantly associated with the occurrence of postsurgical PE (p < 0.0001, AUC 0.80). Patients with CTX had longer durations of both MV (p < 0.0001) and subsequent ventilatory assistance with spontaneous breathing (p = 0.0004), increased total lengths of hospitalization (p < 0.0001), increased durations of ECMO (p < 0.01) and increased incidences of CLD (p < 0.0001) compared to patients without CTX. No significant difference could be found for survival in both groups (p = 0.12). Conclusions: Our data suggest that the incidence of chylothorax is associated with large diaphragmatic defects, the need for postsurgical ECMO and the development of chronic lung disease, but not with survival.

Barriers and prospects for skin grafting in burn treatment across African countries.

INTRODUCTION: The current standard management of full-thickness or deep dermal burns is early tangential excision and skin grafting. A conservative approach to deep burns without the option of skin grafting results in delayed wound healing, possibly leading to wound infection and is associated with hypertrophic scarring and increased morbidity and mortality. The aim of this study was to improve the understanding of the management and availability to perform skin grafting for burns on the African continent. It also sought to identify challenges and perceived improvements. METHODS: A web-based, structured, closed-formatted, multinational survey was designed to gather information on the current state and availability of skin grafting of burn wounds on the African continent. The questionnaire consisted of 27 questions, available in English and French. It was reviewed within the GAP-Burn collaboration network and sent to 271 health care professionals who had participated in a previous study and had initially been recruited by means of the snowball system. RESULTS: The questionnaire was completed 84 times (response rate: 31.0%), of which 3 were excluded. Responses originated from 22 African countries. The majority 71 (87.7%) resulted from countries with a low Human Development Index (HDI), 7 (8.6%) from medium HDI countries. Split thickness skin grafting (STSG) is performed in 51 (63.0%) centers. The majority considers STSG to reduce length of stay (72.8%) and improve scarring (54.3%), yet some indicated that STSG is associated with increased risk of donor site infection (8.6%) and severe bleeding (7.4%). Factors preventing increased grafting included lack of equipment and training. CONCLUSION: Skin grafting is not performed in a significant number of hospitals treating burns. The majority of the staff believe that more skin grafting would lead to a better outcome. Advocacy and improved infrastructure, human resources coupled with introduction to well-structured health coverage for all in African countries could help to better access and affordability in burn care.

Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

PURPOSE: CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. METHODS: Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. RESULTS: We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). CONCLUSION: Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

Fetal MRI-Based Mediastinal Shift Angle (MSA) and Percentage Area of Left Ventricle (pALV) as Prognostic Parameters for Congenital Diaphragmatic Hernia.

OBJECTIVE: Fetal magnetic resonance imaging (MRI) is broadly used as a method for assessing prognosis in congenital diaphragmatic hernia (CDH). In addition to the extent of lung hypoplasia, determined by measuring the lung volume, cardiac impairment due to pulmonary hypertension and left cardiac hypoplasia is decisive for the prognosis. The percentage area of left ventricle (pALV) describes the percentage of the inner area of the left ventricle in relation to the total area, whereas the mediastinal shift angle (MSA) quantifies the extent of cardiac displacement. The prognostic value of pALV and MSA should be evaluated in terms of survival, the need for extracorporeal membrane oxygenation (ECMO) therapy, and the development of chronic lung disease (CLD). METHODS: In a total of 122 fetal MRIs, the MSA and pALV were measured retrospectively and complete outcome parameters were determined regarding survival for all 122 subjects, regarding ECMO therapy in 109 cases and about the development of CLD in 78 cases. The prognostic value regarding the endpoints was evaluated using logistic regression and ROC analysis. RESULTS: The MSA was significantly higher in children who received ECMO therapy (p = 0.0054), as well as in children who developed CLD (p = 0.0018). ROC analysis showed an AUC of 0.68 for ECMO requirement and 0.77 with respect to CLD development. The pALV showed a tendency towards higher levels in children who received ECMO therapy (p = 0.0824). The MSA and the pALV had no significant effect on survival (MSA: p = 0.4293, AUC = 0.56; pALV: p = 0.1134, AUC = 0.57). CONCLUSIONS: The MSA determined in fetal MRI is a suitable prognostic parameter for ECMO requirement and CLD development in CDH patients and can possibly be used as a supplement to the established parameters.

Long-Term Evaluation of the Shape of the Reconstructed Diaphragm in Patients with Left-Sided Congenital Diaphragmatic Hernia Using Serial Chest Radiographs and Correlation to Further Complications.

Background: Defining risk factors for long-term comorbidities in patients after neonatal repair of congenital diaphragmatic hernia (CDH) is an important cornerstone of the implementation of targeted longitudinal follow-up programs. Methods: This study systematically assessed serial chest radiographs of 89 patients with left-sided CDH throughout a mean follow-up of 8.2 years. These geometrical variables for the left and right side were recorded: diaphragmatic angle (LDA, RDA), diaphragmatic diameter (LDD, RDD), diaphragmatic height (LDH, RDH), diaphragmatic curvature index (LDCI, RDCI), lower lung diameter (LLLD, RLLD) and thoracic area (LTA, RTA). Results: It was demonstrated that the shape of the diaphragm in patients with large defects systematically differs from that of patients with small defects. Characteristically, patients with large defects present with a smaller LDCI (5.1 vs. 8.4, p < 0.001) at 6 months of age, which increases over time (11.4 vs. 7.0 at the age of 15.5 years, p = 0.727), representing a flattening of the patch and the attached rudimentary diaphragm as the child grows. Conclusions: Multiple variables during early follow-up were significantly associated with comorbidities such as recurrence, scoliotic curves of the spine and a reduced thoracic area. Some geometrical variables may serve as surrogate parameters for disease severity, which is associated with long-term comorbidities.

Radiomics-Assisted Computed Tomography-Based Analysis to Evaluate Lung Morphology Characteristics after Congenital Diaphragmatic Hernia.

Purpose: Children with congenital diaphragmatic hernia suffer from long-term morbidity, including lung function impairment. Our study aims to analyze lung morphology characteristics via radiomic-assisted extraction of lung features in patients after congenital diaphragmatic hernia repair. Materials and Methods: 72 patients were retrospectively analyzed after approval by the local research ethics committee. All the image data were acquired using a third-generation dual-source CT (SOMATOM Force, Siemens Healthineers, Erlangen, Germany). Dedicated software was used for image analysis, segmentation, and processing. Results: Radiomics analysis of pediatric chest CTs of patients with status after CDH was possible. Between the ipsilateral (side of the defect) and contralateral lung, three shape features and two higher-order texture features were considered statistically significant. Contralateral lungs in patients with and without ECMO treatment showed significant differences in two shape features. Between the ipsilateral lungs in patients with and without the need for ECMO 1, a higher-order texture feature was depicted as statistically significant. Conclusions: By adding quantitative information to the visual assessment of the radiologist, radiomics-assisted feature analysis could become an additional tool in the future to assess the degree of lung hypoplasia in order to further improve the therapy and outcome of CDH patients.

Neutrophil extracellular traps and DNases orchestrate formation of peritoneal adhesions.

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.

Case Report: Fetoscopic Laparoschisis (FETO-LAP)-A New Therapeutic Route to Explore for Fetuses with Severe Diaphragmatic Hernias.

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.

Health-Related Quality of Life and Mental Health of Children with Embryonal Abdominal Tumors.

(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.

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues.

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 µm thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively. Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

Abdominal Wall Movements Predict Intra-Abdominal Pressure Changes in Rats: A Novel Non-Invasive Intra-Abdominal Pressure Detection Method.

(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.

Description of a new clinical syndrome: thoracic constriction without evidence of the typical funnel-shaped depression-the "invisible" pectus excavatum.

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 < 0.01). Strain analysis revealed only discrete changes in RV strain, implying a purely mechanical problem in the absence of structural changes. RV dimensions were negatively correlated with the size of thoracic indices (r = 0.41), reflecting the extent of thoracic constriction. MVP was more prevalent in patients with greater thoracic indices (r = 0.24). The described cohort, referred to as internal PE because of the absence of external changes, showed similar CMR morphologic findings as patients with real PE (especially altered dimensions of the right heart and a lower RVEF). In addition, there was a high incidence of rhythm disturbances, such as extrasystoles or arrhythmias. In one-third of the study cohort additional abnormalities such as pericardial effusion or MVP were present, with MVP being found more frequently in patients with larger thoracic indices, suggesting a possible common pathogenesis.Trial registration: ISRCTN registry, ISRCTN15355937, retrospectively registered 03.06.2022, https://www.isrctn.com/ISRCTN15355937?q=15355937&filters=&sort=&offset=1&totalResults=1&page=1&pageSize=10 .

Expanded ILC2s in human infant intestines promote tissue growth.

Early life is characterized by extraordinary challenges, including rapid tissue growth and immune adaptation to foreign antigens after birth. During this developmental stage, infants have an increased risk of immune-mediated diseases. Here, we demonstrate that tissue-resident, interleukin (IL)-13- and IL-4-producing group 2 innate lymphoid cells (ILC2s) are enriched in human infant intestines compared to adult intestines. Organoid systems were employed to assess the role of infant intestinal ILC2s in intestinal development and showed that IL-13 and IL-4 increased epithelial cell proliferation and skewed cell differentiation toward secretory cells. IL-13 furthermore upregulated the production of mediators of type-2 immunity by infant intestinal epithelial cells, including vascular endothelial growth factor-A and IL-26, a chemoattractant for eosinophils. In line with these in vitro findings increased numbers of eosinophils were detected in vivo in infant intestines. Taken together, ILC2s are enriched in infant intestines and can support intestinal development while inducing an epithelial secretory response associated with type 2 immune-mediated diseases.

Clinical outcome, quality of life, and mental health in long-gap esophageal atresia: comparison of gastric sleeve pull-up and delayed primary anastomosis.

INTRODUCTION: Pediatric surgeons have yet to reach a consensus whether a gastric sleeve pull-up or delayed primary anastomosis for the treatment of esophageal atresia (EA), especially of the long-gap type (LGEA) should be performed. Thus, the aim of this study was to evaluate clinical outcome, quality of life (QoL), and mental health of patients with EA and their parents. METHODS: Clinical outcomes of all children treated with EA from 2007 to 2021 were collected and parents of affected children were asked to participate in questionnaires regarding their Quality of Life (QoL) and their child's Health-Related Quality of Life (HRQoL), as well as mental health. RESULTS: A total of 98 EA patients were included in the study. For analysis, the cohort was divided into two groups: (1) primary versus (2) secondary anastomosis, while the secondary anastomosis group was subdivided into (a) delayed primary anastomosis and (b) gastric sleeve pull-up and compared with each other. When comparing the secondary anastomosis group, significant differences were found between the delayed primary anastomosis and gastric sleeve pull-up group; the duration of anesthesia during anastomosis surgery (478.54 vs 328.82 min, p < 0.001), endoscopic dilatation rate (100% vs 69%, p = 0.03), cumulative time spent in intensive care (42.31 vs 94.75 days, p = 0.03) and the mortality rate (0% vs 31%, p = 0.03). HRQoL and mental health did not differ between any of the groups. CONCLUSION: Delayed primary anastomosis or gastric sleeve pull-up appear to be similar in patients with long-gap esophageal atresia in many key aspects like leakage rate, strictures, re-fistula, tracheomalacia, recurrent infections, thrive or reflux. Moreover, HrQoL was comparable in patients with (a) gastric sleeve pull-up and (b) delayed primary anastomosis. Future studies should focus on the long-term results of either preservation or replacement of the esophagus in children.

The role of neutrophil extracellular traps in necrotizing enterocolitis.

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.

A rapid and convenient sample preparation method for the analysis of cannabinoids in oral fluid samples.

Cannabis is the world's most used illegal drug. The main psychoactive component of cannabis is Δ9-tetrahydrocannabinol (THC). To aid the identification of cannabis-impaired individuals, a simple but effective workflow for reliable quantification of THC and its metabolites in oral fluid samples collected with the Greiner Bio-One Saliva Collection System is presented. Sampling involves rinsing the oral cavity with an extraction solution containing a citrate buffer stimulating salivary flow. Sample processing targeted the cannabinoid fraction interacting with proteins and other insoluble constituents that can be separated by centrifugation. Approximately 50% of the total amount of cannabinoids included in the oral fluid was recovered from the obtained pellet by extraction with acetonitrile. Liquid chromatography-tandem mass spectrometry was used for cannabinoid quantification. Fitness of the developed workflow for application in forensic and clinical cannabis testing was demonstrated by evaluating multiple performance parameters, including selectivity, linearity, limits of quantification (LOQs), accuracy, precision, matrix effects, extraction recoveries, process efficiencies and stability. Furthermore, sensitivity and specificity of the developed oral fluid-based cannabis test was demonstrated by analysing 195 samples collected either from opioid addicts or persons suspected of driving under the influence of drugs. The accuracy of identifying a person with the presence of THC in blood was found to be 97.9%.