Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vß21.3+ activation in multi-inflammatory syndrome in children.

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vß21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vß21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vß21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

Profiling gut microbiota and bile acid metabolism in critically ill children.

Broad-spectrum antimicrobial use during the treatment of critical illness influences gastrointestinal fermentation endpoints, host immune response and metabolic activity including the conversion of primary to secondary bile acids. We previously observed reduced fermentation capacity in the faecal microbiota of critically ill children upon hospital admission. Here, we further explore the timecourse of the relationship between the microbiome and bile acid profile in faecal samples collected from critically ill children. The microbiome was assayed by sequencing of the 16S rRNA gene, and faecal water bile acids were measured by liquid chromatography mass spectrometry. In comparison to admission faecal samples, members of the Lachnospiraceae recovered during the late-acute phase (days 8-10) of hospitalisation. Patients with infections had a lower proportion of Lachnospiraceae in their gut microbiota than controls and patients with primary admitting diagnoses. Keystone species linked to ecological recovery were observed to decline with the length of PICU admission. These species were further suppressed in patients with systemic infection, respiratory failure, and undergoing surgery. Bile acid composition recovers quickly after intervention for critical illness which may be aided by the compositional shift in Lachnospiraceae. Our findings suggest gut microbiota recovery can be readily assessed via measurement of faecal bile acids.

Effect of nutrition status and inflammatory stimuli on ghrelin and peptide-YY levels among critically ill children: A prospective and observational study.

BACKGROUND: Ghrelin and peptide-YY (PYY) are two gut peptides with apparent opposing actions. In normal conditions, ghrelin and PYY work together in synergy to regulate energy homeostasis. During critical illness, series of metabolic, endocrine, and inflammatory changes take place in response to a severe insult. Emerging studies recorded alterations in gut hormone levels in critically ill adults. This study aims to assess the effect of inflammation, nutrition, and feeding status on ghrelin and PYY levels in critically ill children. METHODS: In this prospective study, we collected blood samples from critically ill children on days 2 or 3 of pediatric intensive care unit (PICU) admission for the analysis of serum ghrelin, PYY, and inflammatory markers. Data related to the intake anthropometry, as well as other clinical data, were collected from patients' records. Multiple linear regression analysis was used to identify factors affecting serum levels of these hormones. RESULTS: Forty-two children admitted to the PICU were included in this study. Ghrelin level was influenced by admission nutrition status of the children and age. PYY was influenced by macronutrient intake and age. Inflammatory markers also showed an association with the measured levels of these hormones, with C-reactive protein being positively associated with ghrelin levels and tumor necrosis factor alpha showing a positive association with PYY levels. CONCLUSION: Although ghrelin and PYY have been linked to feeding status in healthy patients, during critical illness there might be other factors, such as inflammation and nutrition status, that might contribute to the changes observed in ghrelin/PYY profiles.

Association between enteral macronutrient delivery and inflammatory response in critically ill children.

BACKGROUND AND AIMS: An important goal of nutrition support in paediatric critical illness is minimising catabolism. While focussing on providing full energy requirements, macronutrient balance is often neglected. Studies suggest that there is interplay between nutrition and inflammation. We aimed to assess the amount of enteral macronutrients delivered compared to estimated requirements, and the association between delivered macronutrients and systemic inflammation in critically ill children. METHOD: We prospectively evaluated energy and macronutrient intake in critically ill children who required at least 72 h of mechanical ventilation. Data on enteral energy and macronutrient intake was collected and expressed as a percentage of the estimated requirements. Circulating levels of inflammatory cytokines were measured by ELISA and association assessed with delivery of macronutrients from the previous 24 h. RESULTS: A total of 87 children (0-16 years) were included in this study. By day 3 the median (IQR) intake of energy, fat, carbohydrate (CHO) and protein were 75% (50-103), 85% (43-120), 63% (42-102) and 45% (23-65) respectively. We have also shown that delivery of enteral fat and protein was associated with elevation in the levels of tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). CONCLUSION: The inflammatory response in critically ill children is influenced by the amount of enteral fat and protein delivered. Our data suggests that within the feed delivered, fat is often higher than protein and CHO. It is crucial to take into account the proportion of macronutrients required and not only aim to achieve the energy goal.

Mesenteric near-infrared spectroscopy and risk of gastrointestinal complications in infants undergoing surgery for congenital heart disease.

We hypothesised that lower mesenteric near-infrared spectroscopy values would be associated with a greater incidence of gastrointestinal complications in children weighing <10 kg who were recovering from cardiac surgery. We evaluated mesenteric near-infrared spectroscopy, central venous oxygen saturation, and arterial blood gases for 48 hours post-operatively. Enteral feeding intake, gastrointestinal complications, and markers of organ dysfunction were monitored for 7 days. A total of 50 children, with median age of 16.7 (3.2-31.6) weeks, were studied. On admission, the average mesenteric near-infrared spectroscopy value was 71±18%, and the systemic oxygen saturation was 93±7.5%. Lower admission mesenteric near-infrared spectroscopy correlated with longer time to establish enteral feeds (r=-0.58, p<0.01) and shorter duration of feeds at 7 days (r=0.48, p<0.01). Children with gastrointestinal complications had significantly lower admission mesenteric near-infrared spectroscopy (58±18% versus 73±17%, p=0.01) and higher mesenteric arteriovenous difference of oxygen at admission [39 (23-47) % versus 19 (4-27) %, p=0.02]. Based on multiple logistic regression, admission mesenteric near-infrared spectroscopy was independently associated with gastrointestinal complications (Odds ratio, 0.95; 95% confidence interval, 0.93-0.97; p=0.03). Admission mesenteric near-infrared spectroscopy showed an area under the receiver operating characteristic curve of 0.76 to identify children who developed gastrointestinal complications, with a suggested cut-off value of 72% (78% sensitivity, 68% specificity). In this pilot study, we conclude that admission mesenteric near-infrared spectroscopy is associated with gastrointestinal complications and enteral feeding tolerance in children after cardiac surgery.

An in vitro model to consider the effect of 2 mM glutamine and KNK437 on endotoxin-stimulated release of heat shock protein 70 and inflammatory mediators.

OBJECTIVE: Glutamine has been shown to promote the release of heat shock protein 70 (HSP70) both within experimental in vitro models of sepsis and in adults with septic shock. This study aimed to investigate the effects of 2 mM glutamine and an inhibitor of HSP70 (KNK437) on the release of HSP70 and inflammatory mediators in healthy adult volunteers. METHODS: An in vitro whole blood endotoxin stimulation assay was used. RESULTS: The addition of 2 mM glutamine significantly increased HSP70 levels over time (P < 0.05). HSP70 release had a positive correlation at 4 h with IL-1 ß (r = 0.51, P = 0.03) and an inverse correlation with TNF-α (r = -0.56, P = 0.02) and IL-8 levels (r = -0.52, P = 0.03), and there were no significant correlations between HSP70 and IL6 or IL-10 or glutamine. Glutamine supplementation significantly (P < 0.05) attenuated the release of IL-10 at 4 h and IL-8 at 24 h, compared with conditions without glutamine. In endotoxin-stimulated blood there were no significant differences in the release of IL-6, TNF-α, and IL-1 ß with glutamine supplementation at 4 and 24 h. However, glutamine supplementation (2 mM) appeared to attenuate the release of inflammatory mediators (IL-1 ß, IL-6, TNF-α), although this effect was not statistically significant. The addition of KNK437, a HSP70 inhibitor, significantly diminished HSP70 release, which resulted in lower levels of inflammatory mediators (P < 0.05). CONCLUSION: Glutamine supplementation promotes HSP70 release in an experimental model of sepsis. After the addition of KNK437, the effects of glutamine on HSP70 and inflammatory mediator release appear to be lost, suggesting that HSP70 in part orchestrates the inflammatory mediator response to sepsis. The clinical implications require further investigation.

Metabolic Profiling of Children Undergoing Surgery for Congenital Heart Disease.

OBJECTIVE: Inflammation and metabolism are closely interlinked. Both undergo significant dysregulation following surgery for congenital heart disease, contributing to organ failure and morbidity. In this study, we combined cytokine and metabolic profiling to examine the effect of postoperative tight glycemic control compared with conventional blood glucose management on metabolic and inflammatory outcomes in children undergoing congenital heart surgery. The aim was to evaluate changes in key metabolites following congenital heart surgery and to examine the potential of metabolic profiling for stratifying patients in terms of expected clinical outcomes. DESIGN: Laboratory and clinical study. SETTING: University Hospital and Laboratory. PATIENTS: Of 28 children undergoing surgery for congenital heart disease, 15 underwent tight glycemic control postoperatively and 13 were treated conventionally. INTERVENTIONS: Metabolic profiling of blood plasma was undertaken using proton nuclear magnetic resonance spectroscopy. A panel of metabolites was measured using a curve-fitting algorithm. Inflammatory cytokines were measured by enzyme-linked immunosorbent assay. The data were assessed with respect to clinical markers of disease severity (Risk Adjusted Congenital heart surgery score-1, Pediatric Logistic Organ Dysfunction, inotrope score, duration of ventilation and pediatric ICU-free days). MEASUREMENTS AND MAIN RESULTS: Changes in metabolic and inflammatory profiles were seen over the time course from surgery to recovery, compared with the preoperative state. Tight glycemic control did not significantly alter the response profile. We identified eight metabolites (3-D-hydroxybutyrate, acetone, acetoacetate, citrate, lactate, creatine, creatinine, and alanine) associated with surgical and disease severity. The strength of proinflammatory response, particularly interleukin-8 and interleukin-6 concentrations, inversely correlated with PICU-free days at 28 days. The interleukin-6/interleukin-10 ratio directly correlated with plasma lactate. CONCLUSIONS: This is the first report on the metabolic response to cardiac surgery in children. Using nuclear magnetic resonance to monitor the patient journey, we identified metabolites whose concentrations and trajectory appeared to be associated with clinical outcome. Metabolic profiling could be useful for patient stratification and directing investigations of clinical interventions.

Nutritional status and clinical outcome in postterm neonates undergoing surgery for congenital heart disease.

OBJECTIVE: Poor growth is a common complication in infants with congenital heart disease. There has been much focus on low birth weight as having increased risk of adverse outcomes following neonatal heart surgery. In this study, we examined whether preoperative nutritional status, measured by admission weight-for-age z score, was associated with postoperative clinical outcome. DESIGN: Retrospective case series. SETTING: Pediatric Cardiac ICU at the Royal Brompton Hospital. PATIENTS: Neonates undergoing surgery for congenital heart disease. Those undergoing ductus arteriosus ligation alone were excluded. Children with coexisting noncardiac morbidity were excluded. Outcome variables included prevalence of postoperative complications (including sepsis, delayed chest closure, renal impairment, and necrotizing enterocolitis), duration of ventilation, intensive care stay, postoperative mortality, and mortality at 1 year after surgery. INTERVENTIONS: None. Analysis of patient data only. MEASUREMENTS AND MAIN RESULTS: Two hundred forty-eight neonates fulfilled the entry criteria. Median (interquartile range) age was 7 days (2-15 d), median (interquartile range) weight was 3.3 kg (2.91-3.6 kg), and median weight-for-age z score was -0.77 (-1.44 to 0.01). Twenty-eight children (11%) had a weight-for-age z score of less than -2. There was no evidence that children with lower weight-for-age z score had less severe surgery as measured by the Risk Adjustment for Congenital Heart Surgery 1 score. In multivariable regression analysis, the weight-for-age z at admission had strong correlation with the number of days free of respiratory support (invasive and noninvasive ventilation) at 28 days (p < 0.0001) and with all-cause mortality at 1 year (p = 0.001). CONCLUSIONS: Poor nutritional status as measured by weight-for-age z is associated with adverse short- and long-term outcomes in neonates undergoing surgery for congenital heart disease.

Intestinal injury and endotoxemia in children undergoing surgery for congenital heart disease.

RATIONALE: Children with congenital heart disease are at risk of gut barrier dysfunction and translocation of gut bacterial antigens into the bloodstream. This may contribute to inflammatory activation and organ dysfunction postoperatively. OBJECTIVES: To investigate the role of intestinal injury and endotoxemia in the pathogenesis of organ dysfunction after surgery for congenital heart disease. METHODS: We analyzed blood levels of intestinal fatty acid binding protein and endotoxin (endotoxin activity assay) alongside global transcriptomic profiling and assays of monocyte endotoxin receptor expression in children undergoing surgery for congenital heart disease. MEASUREMENTS AND MAIN RESULTS: Levels of intestinal fatty acid binding protein and endotoxin were greater in children with duct-dependent cardiac lesions. Endotoxemia was associated with severity of vital organ dysfunction and intensive care stay. We identified activation of pathogen-sensing, antigen-processing, and immune-suppressing pathways at the genomic level postoperatively and down-regulation of pathogen-sensing receptors on circulating immune cells. CONCLUSIONS: Children undergoing surgery for congenital heart disease are at increased risk of intestinal mucosal injury and endotoxemia. Endotoxin activity correlates with a number of outcome variables in this population, and may be used to guide the use of gut-protective strategies.

Myocardial depressant effects of interleukin 6 in meningococcal sepsis are regulated by p38 mitogen-activated protein kinase.

OBJECTIVES: Myocardial failure, leading to inotrope-unresponsive shock, is the predominant cause of death in meningococcal and other forms of septic shock. Proinflammatory cytokines released in septic shock are known to have myocardial depressant effects. We previously showed that interleukin 6 is a major myocardial depressant factor in children with meningococcal septicemia. In the current study, we aimed to investigate the mechanisms by which interleukin 6 induces myocardial failure in meningococcal sepsis and to identify potential novel therapeutic targets. DESIGN: Laboratory-based study. SETTING: University hospital and laboratories. PATIENTS: Children with a clinical diagnosis of meningococcal septic shock. METHODS: We studied interleukin 6-induced signaling events, both in vitro using isolated rat ventricular cardiac myocytes as a model of myocardial contractility and in whole blood from children with meningococcal sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We demonstrated involvement of Janus kinase 2, phosphatidylinositol 3-kinase, Akt, and p38 mitogen-activated protein kinase in interleukin 6-induced negative inotropy in isolated cardiac myocytes. Inhibition of p38 mitogen-activated protein kinase not only reversed interleukin 6-induced myocardial depression in both rat and human myocytes, but restored inotrope responsiveness. Cardiomyocytes transduced with dominant-negative p38 mitogen-activated protein kinase showed no interleukin 6-induced myocardial depression. To investigate p38 mitogen-activated protein kinase in vivo, we profiled global RNA expression patterns in peripheral blood of children with meningococcal septicemia. Transcripts for genes mapping to the p38 mitogen-activated protein kinase pathway showed significantly altered levels of abundance with a high proportion of genes of this pathway affected. CONCLUSIONS: Our findings demonstrate an integral role of the p38 mitogen-activated protein kinase pathway in interleukin 6-mediated cardiac contractile dysfunction and inotrope insensitivity. Dysregulation of the p38 mitogen-activated protein kinase pathway in meningococcal septicemia suggests that this pathway may be an important target for novel therapies to reverse myocardial dysfunction in patients with meningococcal septic shock who are not responsive to inotropic support.

Challenge of predicting resting energy expenditure in children undergoing surgery for congenital heart disease.

OBJECTIVES: To determine pre- and postoperative predictors of energy expenditure in children with congenital heart disease requiring open heart surgery; and to compare measured resting energy expenditure with current predictive equations. DESIGN: Prospective resting energy expenditure data were collected, using indirect calorimetry, for ventilated children admitted consecutively to the pediatric intensive care unit after surgery for congenital heart disease. A 30-min steady-state measurement was performed in suitable patients. Resting energy expenditure was compared to pre- and postoperative clinical variables, and to predicted energy expenditure, using currently used predictive equations. SETTING: Pediatric intensive care unit at the Royal Brompton Hospital, London. PATIENTS: Children ventilated in the pediatric intensive care unit post surgery for congenital heart disease. INTERVENTIONS: Measurement of energy expenditure by indirect calorimetry. MEASUREMENTS AND MAIN RESULTS: Twenty-one mechanically ventilated children (n = 17 boys, 4 girls) were enrolled in the study. Mean +/- sd measured resting energy expenditure was 67.8 +/- 15.4 kcal/kg/day. Most children had inadequate delivery of nutrients compared with actual requirements. Cardiopulmonary bypass had a significant influence on energy expenditure after surgery; in patients who underwent cardiopulmonary bypass during surgery, mean resting energy expenditure was 73.6 +/- 14.45 kcal/kg/day vs. 58.3 +/- 10.29 kcal/kg/day in patients undergoing nonbypass surgery. Children who were malnourished preoperatively had greater resting energy expenditure postoperatively. There was also a significant difference between measured energy expenditure and the Schofield (p = .006), World Health Organization (p = .002), and pediatric intensive care unit-specific formula (p < .0001). However, energy expenditure or a relative energy deficit in the early postoperative period was not associated with severity or duration of organ dysfunction. CONCLUSIONS: Poor nutritional status preoperatively and cardiopulmonary bypass were associated with a greater energy expenditure post cardiac surgery. None of the current predictive equations predicted energy requirements within acceptable clinical accuracy.

Anti-inflammatory modalities: their current use in pediatric cardiac surgery in the United Kingdom and Ireland.

OBJECTIVE: To determine the use of anti-inflammatory therapies in infants and children undergoing cardiac surgery in the United Kingdom and Ireland. DESIGN: Questionnaire survey. SUBJECTS: All centers that undertake pediatric cardiac surgery in the United Kingdom and Ireland. RESULTS: All centers use at least one anti-inflammatory therapy, with 46% of centers using more than one. Both modified ultrafiltration (80%) and steroids (80%) are widely used as anti-inflammatory strategies. Among centers that use steroids, dose, preparation, and timing of steroid administered was highly variable. Heparin-bonded circuits and aprotinin are infrequently used as anti-inflammatory techniques. CONCLUSION: Although anti-inflammatory interventions are believed to contribute to improved patient outcome following cardiopulmonary bypass, this survey has shown that there are still widespread variations in practice. Rather than reflecting poor clinical practice, we believe this reflects a lack of good evidence supporting clinical benefit.

Changes in the interleukin-6/soluble interleukin-6 receptor axis in meningococcal septic shock.

OBJECTIVE: Interleukin-6 is strongly associated with disease severity and outcome in meningococcal septicemia. It is known that interleukin-6 exerts many of its effects via the soluble interleukin-6 receptor. By facilitating the activity of interleukin-6, it is likely that alterations in the levels of soluble interleukin-6 receptor in septic shock could affect the severity of disease. We aimed to investigate changes in the levels of interleukin-6 and soluble interleukin-6 receptor in acute meningococcal septicemia and the relationship with disease severity. DESIGN: Laboratory investigation of interleukin-6 and soluble interleukin-6 receptor levels in children with meningococcal disease. SETTING: University hospital and laboratories. SUBJECTS: Children with severe meningococcal disease requiring intensive care. INTERVENTIONS: Blood samples obtained on admission to the intensive care unit were analyzed for interleukin-6 and soluble interleukin-6 receptor levels. Levels were also serially measured for up to 48 hrs in a subset of patients. MEASUREMENTS AND MAIN RESULTS: Cytokine levels were measured by enzyme-linked immunosorbent assay using mouse monoclonal antihuman antibodies. Acute meningococcemia is associated with a reduction in soluble interleukin-6 receptor levels in proportion to disease severity and is inversely related to interleukin-6 levels. Soluble interleukin-6 receptor returns to levels seen in normal donors following recovery from meningococcal septicemia. CONCLUSIONS: Changes in the levels of interleukin-6 and soluble interleukin-6 receptor in acute meningococcemia may affect the severity and progression of multiple organ failure. Interventions to modulate this axis may improve outcome from meningococcal septic shock.

Role of interleukin 6 in myocardial dysfunction of meningococcal septic shock.

BACKGROUND: Myocardial failure has a central role in the complex pathophysiology of septic shock and contributes to organ failure and death. During the sepsis-induced inflammatory process, specific factors are released that depress myocardial contractile function. We aimed to identify these mediators of myocardial depression in meningococcal septic shock. METHODS: We combined gene-expression profiling with protein and cellular methods to identify a serum factor causing cardiac dysfunction in meningococcal septic shock. We identified genes that were significantly upregulated in blood after exposure to meningococci. We then selected for further analysis those genes whose protein products had properties of a myocardial depressant factor--specifically a 12-25 kDa heat-stable protein that is released into serum shortly after onset of meningococcal infection. FINDINGS: We identified 174 significantly upregulated genes in meningococcus-infected blood: six encoded proteins that were of the predicted size and had characteristics of a myocardial depressant factor. Of these, interleukin 6 caused significant myocardial depression in vitro. Removal of interleukin 6 from serum samples of patients with meningococcaemia and from supernatants of inflammatory cells stimulated by meningococci in vitro abolished the negative inotropic activity. Furthermore, concentrations in serum of interleukin 6 strongly predicted degree of myocardial dysfunction and severity of disease in children with meningococcal septic shock. INTERPRETATION: Interleukin 6 is a mediator of myocardial depression in meningococcal disease. This cytokine and its downstream mediators could be a target for future treatment strategies.

Characterization of a myocardial depressant factor in meningococcal septicemia.

OBJECTIVE: Identification and characterization of myocardial depressant factors present in meningococcal septicemia. DESIGN: Laboratory investigation of myocardial depression that used isolated cardiac myocytes as an model of cardiac contractile function. SETTING: University hospital and laboratories. PATIENTS: Children with severe meningococcal septic shock requiring intensive care. ANIMALS: Myocytes obtained from adult male Sprague-Dawley rats. INTERVENTIONS: Serum samples obtained from the acute phase of sepsis were evaluated for the presence of myocardial depressant activity. Further characterization of the myocardial depressant factor was undertaken by using cell culture supernatants from whole blood and peripheral blood mononuclear cells that had been exposed to heat-killed meningococci. MEASUREMENTS AND MAIN RESULTS: Myocardial depressant activity was measured by using isolated rat left-ventricular myocytes. Changes in amplitude of contraction and in the speed of contraction and relaxation were determined after cells were exposed to various stimuli. Serum from patients with meningococcal disease had myocardial depressant activity. This activity was also present in whole blood and peripheral blood mononuclear cells exposed to meningococci. Myocardial depressant activity was found to be heat stable, proteinaceous, and of a molecular weight range of 10-25 kDa. The activity did not elevate concentrations of cyclic guanylic acid. Lipopolysaccharide-binding protein augmented the release of myocardial depressant factor by peripheral blood mononuclear cells exposed to meningococci. CONCLUSIONS: Myocardial depression in meningococcal sepsis is mediated in part by circulating myocardial depressant factors. Myocardial depressant factors are also released when whole blood or peripheral blood mononuclear cells of healthy donors are exposed to heat-killed meningococci. Release of the factors appears to be mediated through endotoxin-induced activation of peripheral blood mononuclear cells, since lipopolysaccharide-binding protein augments release in a dose-responsive manner. Partial physicochemical characterization of the factors has been achieved.