Immune and metabolic markers for identifying and investigating severe Coronavirus disease and Sepsis in children and young people (pSeP/COVID ChYP study): protocol for a prospective cohort study.

INTRODUCTION: Early recognition and appropriate management of paediatric sepsis are known to improve outcomes. A previous system's biology investigation of the systemic immune response in neonates to sepsis identified immune and metabolic markers that showed high accuracy for detecting bacterial infection. Further gene expression markers have also been reported previously in the paediatric age group for discriminating sepsis from control cases. More recently, specific gene signatures were identified to discriminate between COVID-19 and its associated inflammatory sequelae. Through the current prospective cohort study, we aim to evaluate immune and metabolic blood markers which discriminate between sepses (including COVID-19) from other acute illnesses in critically unwell children and young persons, up to 18 years of age. METHODS AND ANALYSIS: We describe a prospective cohort study for comparing the immune and metabolic whole-blood markers in patients with sepsis, COVID-19 and other illnesses. Clinical phenotyping and blood culture test results will provide a reference standard to evaluate the performance of blood markers from the research sample analysis. Serial sampling of whole blood (50 µL each) will be collected from children admitted to intensive care and with an acute illness to follow time dependent changes in biomarkers. An integrated lipidomics and RNASeq transcriptomics analyses will be conducted to evaluate immune-metabolic networks that discriminate sepsis and COVID-19 from other acute illnesses. This study received approval for deferred consent. ETHICS AND DISSEMINATION: The study has received research ethics committee approval from the Yorkshire and Humber Leeds West Research Ethics Committee 2 (reference 20/YH/0214; IRAS reference 250612). Submission of study results for publication will involve making available all anonymised primary and processed data on public repository sites. TRIAL REGISTRATION NUMBER: NCT04904523.

Late presentations of minor head injury.

OBJECTIVE: To determine the frequency with which a CT head is performed in patients presenting more than 4 h after minor head injury and the clinical features that predict an abnormal scan. DESIGN: Observational cohort study. SETTING: Emergency department (ED) of the Royal London Hospital, London, UK. PARTICIPANTS: 500 patients presenting to the ED of the Royal London Hospital. METHODS: Chart review of all patients with minor head injury presenting more than 4 h from insult to the ED between December 2007 and May 2009. Inclusion criteria were: age over 16 years, Glasgow coma scale (GCS) 14 or 15 on first assessment, over 4 h post-injury. Exclusion criteria were: age under 16 years, GCS 13 and less, injuries limited to the face with no risk of intracranial injury; presentation less than 4 h after injury. RESULTS: 497 patients were identified: 147 patients had CT head; 11 had intracranial injuries on CT. Loss of consciousness (p=0.0005), potential coagulopathy (p=0.0015), injuries above the clavicles (p=0.0150), open/depressed skull fracture (p=0.0221), alcohol/drug intoxication (p=0.0406) and focal neurology (p=0.0562) were predictors of positive CT scan. Five patients (1% of sample, 45% of patients with abnormal CT) required a neurosurgical procedure. Two (18.2%) self-discharged and four (36.4%) were followed up as outpatients. One patient (0.09%) died as a result of intracranial injury. DISCUSSION: Patients with minor head injury who present over 4 h post-insult exhibit a similar risk of intracranial pathology to those presenting within 4 h. The risk factors previously identified to predict intracranial injury are similar in this study.

Leukotriene E4 activates human Th2 cells for exaggerated proinflammatory cytokine production in response to prostaglandin D2.

PGD(2) exerts a number of proinflammatory responses through a high-affinity interaction with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and has been detected at high concentrations at sites of allergic inflammation. Because cysteinyl leukotrienes (cysLTs) are also produced during the allergic response, we investigated the possibility that cysLTs may modulate the response of human Th2 cells to PGD(2). PGD(2) induced concentration-dependent Th2 cytokine production in the absence of TCR stimulation. Leukotrienes D(4) and E(4) (LTE(4)) also stimulated the cytokine production but were much less active than PGD(2). However, when combined with PGD(2), cysLTs caused a greater than additive enhancement of the response, with LTE(4) being most effective in activating Th2 cells. LTE(4) enhanced calcium mobilization in response to PGD(2) in Th2 cells without affecting endogenous PGD(2) production or CRTH2 receptor expression. The effect of LTE(4) was inhibited by montelukast but not by the P2Y(12) antagonist methylthioadenosine 5'-monophosphate. The enhancing effect was also evident with endogenous cysLTs produced from immunologically activated mast cells because inhibition of cysLT action by montelukast or cysLT synthesis by MK886, an inhibitor of 5-lipoxygenase-activating protein, reduced the response of Th2 cells to the levels produced by PGD(2) alone. These findings reveal that cysLTs, in particular LTE(4), have a significant proinflammatory impact on T cells and demonstrate their effects on Th2 cells are mediated by a montelukast-sensitive receptor.

Novel function of CRTH2 in preventing apoptosis of human Th2 cells through activation of the phosphatidylinositol 3-kinase pathway.

It is now well established that interaction of PGD(2) with chemoattractant receptor- homologous molecule expressed on Th2 cells (CRTH2) promotes chemotaxis and proinflammatory cytokine production by Th2 lymphocytes. In this study we show a novel function of CRTH2 in mediating an inhibitory effect of PGD(2) on the apoptosis of human Th2 cells induced by cytokine deprivation. This effect was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD(2), inhibited by the CRTH2 antagonists ramatroban and TM30089, and not observed in CRTH2-negative T cells. D prostanoid receptor 1 (DP(1)) or the thromboxane-like prostanoid (TP) receptor did not play a role in mediating the effects of PGD(2) on the apoptosis of Th2 cells because neither the DP(1) antagonist BW868C nor the TP antagonist SQ29548 had any effect on the antiapoptotic effect of PGD(2). Apoptosis of Th2 cells induced by Fas ligation was not suppressed by treatment with PGD(2), illustrating that activation of CRTH2 only inhibits apoptosis induced by cytokine deprivation. Treatment with PGD(2) induced phosphorylation of Akt and BAD, prevented release of cytochrome c from mitochondria, and suppressed cleavage of caspase-3 and poly(ADP-ribose) polymerase in Th2 cells deprived of IL-2. The PI3K inhibitor LY294002 blocked the effect of PGD(2) both on the signaling events and on the apoptotic death of Th2 cells. These data suggest that in addition to promoting the recruitment and activation of Th2 cells, PGD(2) may also impede the resolution of allergic inflammation through inhibiting apoptosis of Th2 cells.

Inhibition of PI3K and calcineurin suppresses chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)-dependent responses of Th2 lymphocytes to prostaglandin D(2).

Interaction of prostaglandin D2 (PGD2) with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) triggers chemotaxis and pro-inflammatory cytokine production by Th2 lymphocytes. We have investigated the role of inhibitors of various cell-signalling pathways on the responses of human CRTH2+ CD4+ Th2 cells to PGD2. Phosphatidylinositol 3-kinase (PI3K) and Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) pathways were activated by PGD2 in Th2 cells in a CRTH2-dependent manner. Inhibition of the PI3K pathway with LY294002 significantly reduced both PGD2-induced cell migration and cytokine (interleukin-4, interleukin-5 and interleukin-13) production. The inhibitory effect of LY294002 on cell migration is likely to be related to cytoskeleton reorganization as it showed a similar potency on PGD2-induced actin polymerization. The calcineurin inhibitors, tacrolimus (FK506) and cyclosporin A, had no effect on cell migration but completely blocked both cytokine production and the nuclear translocation of NFATc1 suggesting that Ca2+/calcineurin/NFAT is involved in CRTH2-dependent cytokine production but not chemotaxis. The promotion of NFAT nuclear location by PI3K activation may be mediated by negative regulation of glycogen synthase kinase-3beta (GSK3beta), since the PGD2-stimulated increase in phospho-GSK3beta was down-regulated by LY294002, and inhibition of GSK3beta by SB216763 enhanced PGD2-induced Th2 cytokine production and reversed the inhibitory effect of LY294002. These data suggest that PI3K and Ca2+/calcineurin/NFAT signalling pathways are critically involved in pro-inflammatory responses of Th2 cells to PGD2.