Gender and sex hormone effects on neonatal innate immune function.

OBJECTIVES: Scientific evidence provides a widened view of differences in immune response between male and female neonates. The X-chromosome codes for several genes important in the innate immune response and neonatal innate immune cells express receptors for, and are inhibited by, maternal sex hormones. We hypothesized that sex differences in innate immune responses may be present in the neonatal population which may contribute to the increased susceptibility of premature males to sepsis. We aimed to examine the in vitro effect of pro-inflammatory stimuli and hormones in neutrophils and monocytes of male and female neonates, to examine the expression of X-linked genes involved in innate immunity and the miRNA profiles in these populations. METHODS: Preterm infants (n = 21) and term control (n = 19) infants were recruited from the Coombe Women and Infants University Hospital Dublin with ethical approval and explicit consent. The preterm neonates (eight female, 13 male) were recruited with a mean gestation at birth (mean ± SD) of 28 ± 2 weeks and corrected gestation at the time of sampling was 30 + 2.6 weeks. The mean birth weight of preterm neonates was 1084 ± 246 g. Peripheral blood samples were used to analyze immune cell phenotypes, miRNA human panel, and RNA profiles for inflammasome and inflammatory genes. RESULTS: Dividing neutrophil results by sex showed no differences in baseline CD11b between sexes among either term or preterm neonates. Examining monocyte CD11b by sex shows, that at baseline, total and classical monocytes have higher CD11b in preterm females than preterm males. Neutrophil TLR2 did not differ between sexes at baseline or following lipopolysaccharide (LPS) exposure. CD11b expression was higher in preterm male non-classical monocytes following Pam3CSK treatment when compared to females, a finding which is unique to our study. Preterm neonates had higher TLR2 expression at baseline in total monocytes, classical monocytes and non-classical monocytes than term. A sex difference was evident between preterm females and term females in TLR2 expression only. Hormone treatment showed no sex differences and there was no detectable difference between males and females in X-linked gene expression. Two miRNAs, miR-212-3p and miR-218-2-3p had significantly higher expression in preterm female than preterm male neonates. CONCLUSIONS: This study examined immune cell phenotypes and x-linked gene expression in preterm neonates and stratified according to gender. Our findings suggest that the responses of females mature with advancing gestation, whereas male term and preterm neonates have very similar responses. Female preterm neonates have improved monocyte activation than males, which likely reflects improved innate immune function as reflected clinically by their lower risk of sepsis. Dividing results by sex showed changes in preterm and term infants at baseline and following LPS stimulation, a difference which is reflected clinically by infection susceptibility. The sex difference noted is novel and may be limited to the preterm or early neonatal population as TLR2 expression on monocytes of older children does not differ between males and females. The differences shown in female and male innate immune cells likely reflect a superior innate immune defense system in females with sex differences in immune cell maturation. Existing human studies on sex differences in miRNA expression do not include preterm patients, and most frequently use either adult blood or cord blood. Our findings suggest that miRNA profiles are similar in neonates of opposite sexes at term but require further investigation in the preterm population. Our findings, while novel, provide only very limited insights into sex differences in infection susceptibility in the preterm population leaving many areas that require further study. These represent important areas for ongoing clinical and laboratory study and our findings represent an important contribution to exiting literature.

Severe neurological impairment and immune function: altered neutrophils, monocytes, T lymphocytes, and inflammasome activation.

BACKGROUND: Infections cause significant morbidity and mortality in children with Severe Neurological Impairment (SNI). Alterations in immune cell numbers and function in children with neurodisability have been reported. We aimed to characterise neutrophil, monocyte and lymphocyte proportions and activation, at baseline and in response to stimulation with lipopolysaccharide, in children with SNI compared to healthy controls. METHODS: Whole blood samples of children with SNI and controls were incubated in the presence or absence of lipopolysaccharide (10 ng/ml). Monocyte and neutrophil function (Cluster of Differentiation (CD)11b, (TLR)-4 and CD66b expression) and lymphocytes were assessed by flow cytometry. Expression of genes involved in the inflammasome (NLR Family Pyrin Domain Containing(NLRP)-3, Apoptosis-Associated Speck-like protein (ASC) and Interleukin(IL)1ß) were assessed by PCR. RESULTS: Monocytes and CD8+ T cells were lower in children with SNI (n = 14). CD66b, was hyporesponsive and monocyte TLR4 was hyperresponsive to lipopolysaccharide in children with SNI compared to controls (n = 14). NLRP3 expression was higher at baseline and IL1ß expression was not upregulated in response to lipopolysaccharide in children with SNI in contrast to controls. CONCLUSION: We have found significant differences in immune regulation in children with SNI compared to controls which may provide a useful therapeutic target in the future. IMPACT: Children with SNI have reduced monocyte and CD8+ T cells. Neutrophils and monocytes in children with SNI show altered markers of activation in response to lipopolysaccharide. Expression of NLRP3 at the RNA level was higher at baseline in children with SNI. This study adds to the existing literature that children with neurological impairment have altered inflammatory and immune cell responses. This may provide a useful therapeutic target to reduce infection-related morbidity and mortality, and tertiary neurological injury in the future.

Sex differences in neonatal brain injury and inflammation.

Neonatal brain injury and associated inflammation is more common in males. There is a well-recognised difference in incidence and outcome of neonatal encephalopathy according to sex with a pronounced male disadvantage. Neurodevelopmental differences manifest from an early age in infancy with females having a lower incidence of developmental delay and learning difficulties in comparison with males and male sex has consistently been identified as a risk factor for cerebral palsy in epidemiological studies. Important neurobiological differences exist between the sexes with respect to neuronal injury which are especially pronounced in preterm neonates. There are many potential reasons for these sex differences including genetic, immunological and hormonal differences but there are limited studies of neonatal immune response. Animal models with induced neonatal hypoxia have shown various sex differences including an upregulated immune response and increased microglial activation in males. Male sex is recognized to be a risk factor for neonatal hypoxic ischemic encephalopathy (HIE) during the perinatal period and this review discusses in detail the sex differences in brain injury in preterm and term neonates and some of the potential new therapies with possible sex affects.

Innate immune dysregulation in multisystem inflammatory syndrome in children (MIS-C).

MIS-C is a systemic inflammation disorder with poorly characterised immunopathological mechanisms. We compared changes in the systemic immune response in children with MIS-C (n = 12, 5-13 years) to healthy controls (n = 14, 5-15 years). Analysis was done in whole blood treated with LPS. Expression of CD11b and Toll-like receptor-4 (TLR4) in neutrophils and monocytes were analysed by flow cytometry. Serum cytokines (IL-1ß, IL-2, IL-6, IL-8, IL-10, IL-Ira, TNF-α, TNF-ß, IFN-Υ, VEGF, EPO and GM-CSF) and mRNA levels of inflammasome molecules (NLRP3, ASC and IL-1ß) were evaluated. Subpopulations of lymphocytes (CD3+, CD19+, CD56+, CD4+, CD8+, TCR Vδ1+, TCR Vδ2+) were assessed at basal levels. Absolute counts of neutrophils and NLR were high in children with MIS-C while absolute counts of lymphocytes were low. Children with MIS-C had increased levels of IL-6, IL-10, TNF-ß and VEGF serum cytokines at the basal level, and significantly increased TNF-ß post-LPS, compared to controls. IL-1RA and EPO decreased at baseline and post-LPS in MIS-C patients compared to controls. The percentage of CD3+ cells, NK cells and Vδ1 was lower while B cells were higher in children with MIS-C than in controls. Dysregulated immune response in children with MIS-C was evident and may be amenable to immunomodulation.

Melatonin Alters Innate Immune Function in Infants with Neonatal Encephalopathy.

INTRODUCTION: Melatonin has been suggested an adjunctive therapy in neonatal encephalopathy (NE). Melatonin reduces oxidative stress and neutrophil activation; however, the immunological effects in NE have not been studied. METHODS: Infants with NE and neonatal controls were prospectively recruited. Whole blood was sampled in the first week of life. Following endotoxin and or melatonin treatment, diurnal variation was measured by RT PCR for circadian rhythm genes (brain and Muscle Arnt-Like protein [BMAL1], circadian locomotor output cycles kaput [CLOCK], Nuclear Receptor Subfamily 1 Group D Member 2 [REV Erß], and cryptochrome circadian clock [CRY]). Neutrophil and monocyte cell surface markers of activation CD11b, reactive oxygen intermediates (ROIs), and Toll-like receptor (TLR)-4 were also examined by flow cytometry in matching samples. RESULTS: Serum and RNA samples from forty infants were included (controls n = 20; NE n = 20) over the first week of life. Melatonin reduced neutrophil CD11b and TLR-4 expression in response to LPS in infants with NE compared to controls. There were no differences in ROIs. BMAL1 and CLOCK baseline gene expression levels were similar. BMAL1 was significantly decreased with LPS stimulation in NE. There was no significant diurnal variation in melatonin, neutrophil, and monocyte function or circadian genes. CONCLUSIONS: Melatonin alters immune function ex vivo in infants with NE. Infants with NE have altered immune circadian responses following LPS stimulation, which have potential for modulation.

Selective modulation of monocyte and neutrophil responses with activated protein C in preterm infants.

BACKGROUND: Inflammation is associated with many disorders of preterm infants including periventricular leukomalacia, chronic lung disease, and necrotizing enterocolitis. Activated protein c (APC) has shown positive immunomodulatory effects. OBJECTIVES: We aimed to study neutrophil and monocyte function in response to lipopolysaccharide (LPS) and APC stimulation ex vivo in preterm infants <32 weeks gestation over the first week of life compared to neonatal and adult controls. METHODS: Peripheral blood was taken on day 1, 3, and 7 and stimulated with LPS in the absence or presence of APC. Expression of toll-like receptor 4 (TLR4) and CD11b and reactive oxygen intermediate (ROI) release from neutrophils and monocytes was examined by flow cytometry. RESULTS: LPS induced neutrophil ROI in adults and preterm infants and was significantly reduced by APC. Baseline and LPS-induced monocyte ROI production in preterm neonates was increased compared to adult and term controls. Neutrophil TLR4 baseline expression was higher in term controls compared to preterm infants. CONCLUSION: Increased systemic ROI release in preterm infants may mediate tissue damage, ROI was reduced by APC. However, due to the high risk of hemorrhage further examination of APC mutant forms with anti-inflammatory but decreased anticoagulant properties is merited.

Infants with Down syndrome and congenital heart disease have altered peri-operative immune responses.

BACKGROUND: Infants with Down syndrome (DS) have an altered immune response. We aimed to characterise the inflammatory response in infants with DS and congenital heart disease (CHD) peri-operatively in comparison to infants with CHD and a normal chromosomal complement, and to healthy infants pre-operatively. METHODS: Infants with DS/CHD, infants without DS but with CHD (CHD only) and healthy infants were prospectively recruited and serial serum cytokines evaluated peri-operatively using multiplex ELISA: tumour necrosis factor (TNF)-α and TNF-ß; interferon (IFN)-γ, interleukin (IL)-1α, IL-2, IL-6, IL-8, IL-18, IL-1ß, IL-10, and IL-1ra; vascular endothelial growth factor (VEGF); granulocyte macrophage colony-stimulating factor (GM-CSF); and erythropoietin (EPO). RESULTS: Ninety-four infants were recruited including age-matched controls (n = 10), DS/CHD (n = 55), and CHD only (n = 29). Children with DS/CHD had significantly lower concentrations of several cytokines (IL-10, IL-6, IL-8, IL-1ß, VEGF) in the pre- and post-operatively vs CHD only and controls. EPO and GM-CSF were significantly higher in DS/CHD (p value <0.05). CONCLUSIONS: Children with DS/CHD had significantly lower concentrations of several cytokines compared to controls or children with CHD only. EPO and GM-CSF were significantly higher in children with DS/CHD. The assessment of the immune response may be suitable for the predictable clinical outcomes in these children. IMPACT: This study demonstrated that children with Down syndrome (DS) and congenital heart disease (CHD) have significant alterations in pro-inflammatory and anti-inflammatory immune responses peri-operatively. These changes may contribute to adverse clinical outcomes, including sepsis, chylothorax, and autoimmunity. They may impact the pathogenesis and outcome post-operatively and long term in this population. Children with DS and CHD have significantly lower cytokine concentrations, increased EPO and GM-CSF, and decreased VEGF pre- and post-operatively. Assessing their inflammatory state peri-operatively may facilitate the development of a predictive model that can inform tailored management of these infants using novel therapies including immunomodulation.

Mild-to-severe traumatic brain injury in children: altered cytokines reflect severity.

BACKGROUND: Paediatric traumatic brain injury (TBI) is recognised to have significant longer-term neurocognitive effects. Childhood is a time of high risk for head injury. Functional recovery is variable with a combination of any or all of physical, cognitive and emotional impairment. Immune activation and alteration in cytokine levels are present following TBI which may differ from adults. METHODS: Pro- and anti-inflammatory cytokines including Interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-17A, Tumor Necrosis Factor (TNF)-α and Interferon (IFN)-γ were examined at baseline and following in vitro treatment with endotoxin of whole blood, in the following children: severe TBI (sTBI: initial Glasgow coma scale(GCS) ≤ 8), mild TBI (mTBI; GCS 14/15) at 0-4d and at 10-14d post-TBI and compared to healthy age-matched controls. RESULTS: The study enrolled 208 children, including 110 with TBI cohort (n = 104 mild; 6 severe) and controls (n = 98). At baseline all children with TBI had increased IL-6. The mTBI group had significantly increased IFN-γ versus controls. In sTBI at baseline, IFN-γ was decreased compared to controls. At baseline IL-8, IL-10, IL-17A, and TNF-α were decreased in mTBI compared to controls. This persisted at 2 week post-mTBI. The AUC for detecting mTBI was 0.801 CI (0.73-086) using IL6/IL10 ratio. mTBI showed a greater fold change in IL-8 and TNF-α in response to endotoxin stimulation, a response that persisted at 2 weeks. Children with sTBI did not have a significant IL-6 response to endotoxin, but did show an increase in IL-17A. CONCLUSION: Children with all TBI including mTBI show altered cytokine profiles and altered endotoxin responses. Although cytokines increased in sTBI especially in response to endotoxin, suppressed responses were found in mTBI coupled with persistent immune dysfunction post-injury.

Traumatic Brain Injury in Children: Glial fibrillary Acidic Protein and Clinical Outcomes.

OBJECTIVES: Glial fibrillary acidic protein (GFAP) is a neuronal protein released after traumatic brain injury (TBI) and detectable in serum samples. GFAP correlates with symptom severity in adults and may be a marker of brain injury in children with milder symptoms or preverbal children. METHODS: GFAP was examined in children with severe TBI (initial Glasgow Coma Scale score <8), with mild TBI (Glasgow Coma Scale score 14/15), and at 0 to 4 and at 10 to 14 days after TBI and was compared with healthy age-matched controls. Mechanism, time points from injury, and symptoms were recorded. RESULTS: The study enrolled 208 children including 110 with TBI (n = 104 mild, 6 severe) and controls (n = 98). GFAP was higher in mild TBI than in controls and highest in the severe TBI cohort, with a maximum value at 6 hours from injury. Vomiting was significantly associated with higher GFAP levels, but no association was found with amnesia, loss of consciousness, and the Sports Concussion Assessment Tool. Children reporting >1-point changes from their preinjury functioning on the Post-Concussive Symptom Inventory had higher initial GFAP but not total Post-Concussive Symptom Inventory score changes. CONCLUSIONS: GFAP identifies children with TBI, even at the milder end of the spectrum, and is strongly associated with postinjury vomiting. It may be a useful marker of pediatric TBI; however, sampling is time critical.

Neonatal encephalopathy plasma metabolites are associated with neurodevelopmental outcomes.

BACKGROUND: To investigate mechanisms of injury and recovery in neonatal encephalopathy (NE), we performed targeted metabolomic analysis of plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS) from healthy term neonates or neonates with NE. METHODS: Plasma samples from the NE (n = 45, day of life 0-1) or healthy neonatal (n = 30, ≥36 weeks gestation) cohorts had LC/MS/MS metabolomic profiling with a 193-plex targeted metabolite assay covering >366 metabolic pathways. Metabolite levels were compared to 2-year neurodevelopmental outcomes measured by the Bayley Scales of Infant and Toddler Development III (Bayley-III). RESULTS: Out of 193 metabolites, 57 met the pre-defined quality control criteria for analysis. Significant (after false discovery rate correction) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways included aminoacyl-tRNA biosynthesis, arginine biosynthesis, and metabolism of multiple amino acids. Significant disease pathways included seizures. In regression models, histidine and C6 sugar amine were significantly associated with cognitive, motor, and language and betaine with cognitive and motor Bayley-III composite scores. The addition of histidine, C6 sugar amine, and betaine to a Sarnat score-based clinical regression model significantly improved model performance (Akaike information criterion and adjusted r2) for Bayley-III cognitive, motor, and language scores. CONCLUSIONS: Plasma metabolites may help to predict neurological outcomes in neonatal brain injury and enhance current clinical predictors. IMPACT: Plasma metabolites may help to predict neurological outcomes in NE and supplement current clinical predictors. Current metabolomics research is limited in terms of clinical application and association with long-term outcomes. Our study presents novel associations of plasma metabolites from the first 24 h of life and 2-year neurodevelopmental outcomes for infants with NE. Our metabolomics discovery provides insight into possible disease mechanisms and methods to rescue and/or supplement metabolic pathways involved in NE. Our metabolomics discovery of metabolic pathway supplementations and/or rescue mechanisms may serve as adjunctive therapies for NE.

Multi-organ dysfunction scoring in neonatal encephalopathy (MODE Score) and neurodevelopmental outcomes.

AIM: Neonatal encephalopathy (NE) is associated with an increased risk of multi-organ injury. The lack of standardised definitions for multi-organ dysfunction in NE hinders accurate quantification of these complications. METHODS: A simple multi-organ dysfunction in neonatal encephalopathy scoring (MODE) system was created to include the cardiovascular, respiratory, gastrointestinal, haematological and neurological systems with a maximum score of 15. The MODE score was then compared with the grade of NE, Bayley Scales of Infant Development (Bayley-III) at 2 years of age and mortality. The Bayley score was used as it gave an objective score making it easier to compare the MODE score. Bayley score of <90 and/or abnormal MRI as an adverse outcome. RESULTS: Infants with perinatal asphyxia (PA:n = 85) were prospectively enrolled (PA only n = 9; NE I = 23; NE II = 42; NE III = 11). Infants with higher MODE scores were significantly more likely to have moderate/severe NE (NE II/III: median scores (IQR) 7(5-10) versus mild NE 2 (1-3); p-value < 0.001) The MODE score was highly predictive of mortality (AUC 0.96, p-value = 0.002). Infants who had an abnormal neurological examination at discharge or abnormal Bayley-III scores had significantly higher MODE scores (p-value = 0.001). CONCLUSION: Quantifying multi-organ injury is important to plan optimal early management and long-term follow-up. Additional use of clinical biomarkers may be useful as surrogate endpoints in future clinical trials and link to multi-organ longer-term developmental follow-up.

Melatonin as an immunomodulator in children with Down syndrome.

BACKGROUND: Down syndrome (DS) is a disorder characterised by marked immune dysfunction, increased mortality from sepsis, chronic inflammation, increased oxidative stress, sleep disturbance and possibly abnormal endogenous melatonin levels. Melatonin has a myriad of immune functions, and we hypothesised that this therapeutic agent could modulate the innate immune system in this cohort. METHODS: We investigated neutrophil and monocyte function (CD11b, TLR4 expression by flow cytometry), genes involved in TLR signalling (MyD88, IRAK4, TRIF), the inflammasome (NLRP3, IL-1ß), and circadian rhythm (BMAL, CLOCK, CRY) by qPCR, and inflammatory cytokines (IL-2, IL-6, IL-8, IL-18, IL-1ß, TNF-α, IFN-γ, IL-10, IL-1ra, VEGF, Epo, GM-CSF) by enzyme-linked immunosorbent assay (ELISA) following immunomodulation with LPS endotoxin and melatonin. 47 children with DS and 23 age- and sex-matched controls were recruited. RESULTS: We demonstrated that melatonin has several significant effects by reducing CD11b and TLR4 expression, attenuating TLR signalling, genes involved in the inflammasome and has the potential to reduce LPS-induced inflammatory responses. CONCLUSIONS: Immunomodulatory effects of melatonin were found in both paediatric cohorts with more marked effects in the children with DS. Melatonin mediates immune response through a wide array of mechanisms and this immunomodulator may buffer the inflammatory response by regulating pro and anti-inflammatory signalling. IMPACT: We highlight that melatonin mediates its immune response through a wide array of mechanisms, its effects appear to be dose dependant and children with Down syndrome may be more receptive to treatment with it. Immunomodulatory effects of melatonin were demonstrated with marked effects in the children with Down syndrome with a reduction of MyD88, IL-1ß and NLRP3 expression in whole-blood samples. Melatonin is a proposed anti-inflammatory agent with a well-established safety profile, that has the potential for mitigation of pro- and anti-inflammatory cytokines in paediatric Down syndrome cohorts, though further clinical trials are warranted.

Relationship Between MRI Scoring Systems and Neurodevelopmental Outcome at Two Years in Infants With Neonatal Encephalopathy.

BACKGROUND: Magnetic resonance imaging (MRI) scoring systems are used in the neonatal period to predict outcome in infants with neonatal encephalopathy. Our aim was to assess the relationship between three MRI scores and neurodevelopmental outcome assessed using Bayley Scales of Infant and Toddler Development, third edition (Bayley-III), at two years in infants with neonatal encephalopathy. METHODS: Term-born neonates with evidence of perinatal asphyxia born between 2011 and 2015 were retrospectively reviewed. MRI scanning was performed within the first two weeks of life and scored using Barkovich, National Institute of Child Health and Human Development (NICHD) Neonatal Research Network (NRN), and Weeke systems by a single assessor blinded to the infants clinical course. Neurodevelopmental outcome was assessed using composite scores on the Bayley-III at two years. Multiple linear regression analyses were used to assess the association between MRI scores and Bayley-III composite scores, with postmenstrual age at scan and sex included as covariates. RESULTS: Of the 135 recruited infants, 90 infants underwent MRI, and of these, 66 returned for follow-up. MRI abnormalities were detected with the highest frequency using the Weeke score (Barkovich 40%, NICHD NRN 50%, Weeke 77%). The inter-rater agreement was good for the Barkovich score and excellent for NICHD NRN and Weeke scores. There was a significant association between Barkovich, NICHD NRN, and Weeke scores and Bayley-III cognitive and motor scores. Only the Weeke score was associated with Bayley-III language scores. CONCLUSIONS: Our findings confirm the predictive value of existing MRI scoring systems for cognitive and motor outcome and suggest that more detailed scoring systems have predictive value for language outcome.

Altered Cytokine Endotoxin Responses in Neonatal Encephalopathy Predict MRI Outcomes.

Background: Neonatal encephalopathy (NE) is associated with adverse neurodevelopmental outcome and is linked with systemic inflammation. Pro-inflammatory and anti-inflammatory cytokines are known to play a role in the pathology of NE by activating innate immune cells. Methods: Eighty-seven infants were enrolled including 53 infants with NE of whom 52 received therapeutic hypothermia (TH) and 34 term infant healthy controls (TC). Whole blood sampling was performed in the first 4 days of life, and a 14-spot ELISA Multiplex Cytokine Array was carried out on baseline samples or after stimulation with lipopolysaccharide (LPS) as an additional inflammatory stimulus. The cytokine medians were examined for differences between infants with NE and healthy TC; and then short-term outcomes of Sarnat stage, seizures, and MRI brain were examined within the NE group. The potential of LPS stimulation to predict abnormal MRI was explored using receiver operating characteristic (ROC) curves. Results: At baseline, infants with NE had significantly higher levels of erythropoietin (Epo), interleukin (IL)-6, and IL-1ra and significantly lower vascular endothelial growth factor (VEGF) than had controls. All cytokines were increased after LPS stimulation in infants with NE with an excessive Epo and IL-1ra response than in controls. Infants with NE had lower IL-8, IL-2, IL-6, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), VEGF, and interferon (IFN)-γ than controls had following LPS. GM-CSF and IFN-γ, IL-1ß, IL-1ra, and VEGF were higher on days 1-2 in NE infants with abnormal neuroimaging. GM-CSF, IFN-γ, and TNF-α levels with LPS stimulation were different upon stimulation between normal and abnormal neuroimaging. TNF-α is the only strong cytokine predictor both pre- and post-LPS stimulation of abnormal brain imaging. Conclusions: Altered cytokine responses are found in infants with NE vs. controls, and more significant differences are unmasked by the additional stimulus of LPS, which potentially improves the predictive power of these cytokines for the detection of abnormal MRIs. Infants with NE undergoing TH demonstrate both trained immunity and tolerance, and understanding these responses will facilitate adjunctive immunomodulatory treatments.

Novel Point-of-Care Diagnostic Method for Neonatal Encephalopathy Using Purine Nucleosides.

Background: Evidence suggests that earlier diagnosis and initiation of treatment immediately after birth is critical for improved neurodevelopmental outcomes following neonatal encephalopathy (NE). Current diagnostic tests are, however, mainly restricted to clinical diagnosis with no molecular tests available. Purines including adenosine are released during brain injury such as hypoxia and are also present in biofluids. Whether blood purine changes can be used to diagnose NE has not been investigated to date. Methods: Blood purines were measured in a mouse model of neonatal hypoxia and infants with NE using a novel point-of-care diagnostic technology (SMARTChip) based on the summated electrochemical detection of adenosine and adenosine metabolites in the blood. Results: Blood purine concentrations were ∼2-3-fold elevated following hypoxia in mice [2.77 ± 0.48 µM (Control) vs. 7.57 ± 1.41 µM (post-hypoxia), p = 0.029]. Data in infants with NE had a 2-3-fold elevation when compared to healthy controls [1.63 ± 0.47 µM (Control, N = 5) vs. 4.87 ± 0.92 µM (NE, N = 21), p = 0.0155]. ROC curve analysis demonstrates a high sensitivity (81%) and specificity (80%) for our approach to identify infants with NE. Moreover, blood purine concentrations were higher in infants with NE and seizures [8.13 ± 3.23 µM (with seizures, N = 5) vs. 3.86 ± 0.56 µM (without seizures, N = 16), p = 0.044]. Conclusion: Our data provides the proof-of-concept that measurement of blood purine concentrations via SMARTChip technology may offer a low-volume bedside test to support a rapid diagnosis of NE.

Neutrophils: Need for Standardized Nomenclature.

Neutrophils are the most abundant innate immune cell with critical anti-microbial functions. Since the discovery of granulocytes at the end of the nineteenth century, the cells have been given many names including phagocytes, polymorphonuclear neutrophils (PMN), granulocytic myeloid derived suppressor cells (G-MDSC), low density neutrophils (LDN) and tumor associated neutrophils (TANS). This lack of standardized nomenclature for neutrophils suggest that biologically distinct populations of neutrophils exist, particularly in disease, when in fact these may simply be a manifestation of the plasticity of the neutrophil as opposed to unique populations. In this review, we profile the surface markers and granule expression of each stage of granulopoiesis to offer insight into how each stage of maturity may be identified. We also highlight the remarkable surface marker expression profiles between the supposed neutrophil populations.

Altered distributions and functions of natural killer T cells and γδ T cells in neonates with neonatal encephalopathy, in school-age children at follow-up, and in children with cerebral palsy.

We enumerated conventional and innate lymphocyte populations in neonates with neonatal encephalopathy (NE), school-age children post-NE, children with cerebral palsy and age-matched controls. Using flow cytometry, we demonstrate alterations in circulating T, B and natural killer cell numbers. Invariant natural killer T cell and Vδ2+ γδ T cell numbers and frequencies were strikingly higher in neonates with NE, children post-NE and children with cerebral palsy compared to age-matched controls, whereas mucosal-associated invariant T cells and Vδ1 T cells were depleted from children with cerebral palsy. Upon stimulation ex vivo, T cells, natural killer cells and Vδ2 T cells from neonates with NE more readily produced inflammatory cytokines than their counterparts from healthy neonates, suggesting that they were previously primed or activated. Thus, innate and conventional lymphocytes are numerically and functionally altered in neonates with NE and these changes may persist into school-age.

An observational study of sleep in childhood post-neonatal encephalopathy.

AIM: Neonatal encephalopathy (NE) is associated with altered cognitive, motor, sensory abilities and behavioural outcomes. This case-control study aimed to assess whether Quality of Life (QoL) and sleep disorders are affected in older children following NE compared to age-matched controls. METHODS: Children at school-age post-NE were recruited and compared to age-matched controls. Sleep and QoL were assessed with the Pediatric Quality of Life Inventory and the Child Sleep Habit Questionnaire. RESULTS: One hundred children were recruited with an age range of 4-6 years, including children post-NE (n=45) and age-matched controls (n = 55). Significantly higher pathological sleep scores were evident in 58% of children post-NE compared to controls (43.8 vs 40.2; p = 0.001). Children post-NE had increased bedtime resistance (p = 0.028) and sleep anxiety (p = 0.01) compared to controls. Children in the post-NE group had lower total QoL scores versus controls (mean score 82.5 vs 95.8; p < 0.01). Children with mild NE also had lower total QoL scores than controls (90.0 vs 95.8, p = 0.003). There was a strong correlation between low QoL with high total sleep scores (Rho 0.339, p = 0.014). CONCLUSION: There were high rates of sleep issues in school-aged children with mild and moderate-severe NE. Consideration and management of sleep problems may improve QoL in childhood post-NE.

Correlation between Medication Administration-Related Errors in Patients with Parkinson Disease and Timing of Pharmacy-Led Best Possible Medication Histories.

BACKGROUND: Poor prescribing and incomplete medication administration have been linked to increased lengths of hospitalization for patients with Parkinson disease. The Institute for Safe Medication Practices (ISMP) has recommended that patients with Parkinson disease receive a pharmacy consultation within 2 h of admission to hospital. OBJECTIVES: To examine whether the time for a pharmacy team member to obtain a best possible medication history (BPMH) was associated with administration-related medication errors. The primary outcome was the proportion of doses with a medication error during a patient's admission in relation to the time to completion of the initial BPMH by a registered pharmacist (RPh) or registered pharmacy technician (RPhT). The secondary objective was to compare the proportion of doses with a medication error in relation to whether the BPMH was completed by an RPh or an RPhT. METHODS: This retrospective chart review involved patients with Parkinson disease who were admitted to the medicine services at London Health Sciences Centre from September 30, 2014, to September 30, 2018. Patients were included if they had Parkinson disease and a medication regimen that included levodopa-carbidopa. For all patients, an RPhT or RPh conducted the initial BPMH or updated the BPMH. Pearson correlation analysis was used to determine whether a correlation existed between administration-related errors and completion of the BPMH by a pharmacy staff member. RESULTS: A total of 84 patients with 104 admissions were included. There was no significant correlation between the time to completion of the initial BPMH by a pharmacy team member and the proportion of doses with medication errors (p = 0.32). Although RPhTs completed the BPMHs more quickly than RPhs (p < 0.001), there was no significant difference between pharmacy team members in terms of the proportion of doses with medication errors (p = 0.86). CONCLUSIONS: Completing a BPMH within 2 h of a patient's admission, as per the ISMP recommendation, is unlikely to affect administration-related medication errors, given that no correlation was identified. Expediting BPMH without addressing other factors is insufficient, and initiatives are required to improve the medication administration process.

CONTEXTE: La mauvaise prescription et l'administration incomplète de médicaments ont été liées à une augmentation de la durée du séjour à l'hôpital des patients atteints de la maladie de Parkinson. L'Institute for Safe Medication Practices (ISMP) a recommandé que les patients atteints de la maladie de Parkinson obtiennent une consultation en pharmacie dans les 2 heures après leur admission à l'hôpital. OBJECTIFS: Vérifier si le temps d'attente pour l'obtention, par un membre de l'équipe de la pharmacie, du meilleur schéma thérapeutique possible (MSTP) était associé ou non à des erreurs de médication liées à l'administration. Le résultat principal portait sur la proportion des doses comportant une erreur de médication lors de l'admission d'un patient par rapport au temps nécessaire à un pharmacien ou à un technicien en pharmacie autorisés pour réaliser le MSTP initial. L'objectif secondaire visait à comparer la proportion des doses comportant une erreur de médication entre un MSTP réalisé par un pharmacien autorisé et un MSTP réalisé par un technicien en pharmacie autorisé. MÉTHODES: Cet examen rétrospectif des dossiers impliquait des patients atteints de la maladie de Parkinson ayant été admis aux services de médecine au London Health Sciences Centre entre le 30 septembre 2014 et le 30 septembre 2018. Les patients pouvaient participer à l'étude s'ils avaient la maladie de Parkinson et qu'ils suivaient un traitement médicamenteux comprenant du lévodopa-carbidopa. Un pharmacien autorisé ou un technicien en pharmacie autorisé réalisait ou actualisait le MSTP initial de tous les patients. La corrélation de Pearson a servi à déterminer s'il existait une corrélation entre les erreurs liées à l'administration et la réalisation du MSTP par un membre du personnel de la pharmacie. RÉSULTATS: Au total, 84 patients correspondant à 104 admissions ont été inclus dans l'étude. Il n'y avait aucune corrélation importante entre le moment de la réalisation du MSTP initial par un membre du personnel de la pharmacie et la proportion des doses comportant des erreurs de médication (p = 0,32). Bien que les techniciens en pharmacie autorisés aient terminé plus rapidement leur MSTP que les pharmaciens autorisés (p < 0,001), aucune différence importante n'a été notée entre les membres du personnel de la pharmacie en termes de proportion des doses et d'erreur de médication (p = 0,86). CONCLUSIONS: Il est peu probable que la réalisation d'un MSTP dans les 2 heures après l'admission d'un patient, conformément à la recommandation de l'ISMP, ait une influence sur les erreurs de médication liées à l'administration, vu qu'aucune corrélation n'a été décelée. Précipiter la réalisation du MSTP sans aborder les autres facteurs ne suffit pas et des actions sont nécessaires pour améliorer le processus d'administration des médicaments.

Cytokine dysregulation in children with cerebral palsy.

AIM: To examine pro- and anti-inflammatory cytokines in children with cerebral palsy (CP) at baseline and in response to endotoxin (lipopolysaccharide), and correlate outcomes compared with age-matched comparisons, to evaluate their ability to mount an immune response. METHOD: Serum cytokines were assessed in 12 children (eight males, four females; mean age 10y 1mo [SD 1y 8mo], 6-16y) with CP against 12 age-matched comparisons (eight males, four females; mean age 9y 1mo [SD 1y 1mo]). Pro- and anti-inflammatory cytokines (interleukin-1ß, interleukin-2, interleukin-6, interleukin-8, interleukin-10, interleukin-18, tumour necrosis factor [TNF]-α, TNF-ß, interferon-γ, granulocyte-macrophage colony-stimulating factor [GM-CSF], vascular endothelial growth factor [VEGF], erythropoietin, and interleukin-1 receptor antagonist) were measured at baseline and in response to in vitro simulation with lipopolysaccharide by multiplex enzyme-linked immunosorbent assay. RESULTS: Significantly higher erythropoietin was found at baseline in children with CP compared with the comparison group. There was a strong response to lipopolysaccharide for interleukin-8, VEGF, TNF-α, and GM-CSF in both children with CP and the comparison group; however, there was significant lipopolysaccharide hyporesponsiveness in children with CP compared with the comparison group for interleukin-1α, interleukin-1ß, interleukin-2, and interleukin-6. INTERPRETATION: Altered cytokine responses in children with CP compared with the comparison group demonstrate an altered inflammatory state that may contribute to ongoing sequelae and could be a target for therapy. WHAT THIS PAPER ADDS: Altered inflammatory responses persist in children with cerebral palsy (CP). Erythropoietin is elevated in children with CP compared with the comparison group. Children with CP have reduced interleukin-1α, interleukin-1ß, interleukin-2, and interleukin-6 inflammatory responses to lipopolysaccharide.