Angiogenesis-associated pathways play critical roles in neonatal sepsis outcomes.

Neonatal sepsis is a major cause of childhood mortality. Limited diagnostic tools and mechanistic insights have hampered our abilities to develop prophylactic or therapeutic interventions. Biomarkers in human neonatal sepsis have been repeatedly identified as associated with dysregulation of angiopoietin signaling and altered arachidonic acid metabolism. We here provide the mechanistic evidence in support of the relevance for these observations. Angiopoetin-1 (Ang-1), which promotes vascular integrity, was decreased in blood plasma of human and murine septic newborns. In preclinical models, administration of Ang-1 provided prophylactic protection from septic death. Arachidonic acid metabolism appears to be functionally connected to Ang-1 via reactive oxygen species (ROS) with a direct role of nitric oxide (NO). Strengthening this intersection via oral administration of arachidonic acid and/or the NO donor L-arginine provided prophylactic as well as therapeutic protection from septic death while also increasing plasma Ang-1 levels among septic newborns. Our data highlight that targeting angiogenesis-associated pathways with interventions that increase Ang-1 activity directly or indirectly through ROS/eNOS provide promising avenues to prevent and/or treat severe neonatal sepsis.

Skin-Microbiome Assembly in Preterm Infants during the First Three Weeks of Life and Impact of Topical Coconut Oil Application.

The structure and function of infant skin is not fully developed until 34 weeks of gestation, and this immaturity is associated with risk of late-onset sepsis (LOS). Topical coconut oil improves preterm-infant skin integrity and may reduce LOS. However, data on early-life skin-microbiome succession and potential effects of emollient skin care in preterm infants are scarce. We therefore collected skin-microbiome samples from the ear, axilla, and groin on days 1, 7, 14, and 21 from preterm infants born <30 weeks of gestation as part of a randomized clinical trial of standard skin care vs. topical coconut oil. We found that within-sample microbiome diversity was highest on day 1 after birth, with a subsequent decline and emergence of Staphylococcus genus dominance from day 7. Moreover, microbiome assembly was less diverse in infants receiving coconut oil vs. standard skin care. Our study provides novel data on preterm-infant skin-microbiome composition and highlights the modifying potential of emollient skin care.

Gene filtering strategies for machine learning guided biomarker discovery using neonatal sepsis RNA-seq data.

Machine learning (ML) algorithms are powerful tools that are increasingly being used for sepsis biomarker discovery in RNA-Seq data. RNA-Seq datasets contain multiple sources and types of noise (operator, technical and non-systematic) that may bias ML classification. Normalisation and independent gene filtering approaches described in RNA-Seq workflows account for some of this variability and are typically only targeted at differential expression analysis rather than ML applications. Pre-processing normalisation steps significantly reduce the number of variables in the data and thereby increase the power of statistical testing, but can potentially discard valuable and insightful classification features. A systematic assessment of applying transcript level filtering on the robustness and stability of ML based RNA-seq classification remains to be fully explored. In this report we examine the impact of filtering out low count transcripts and those with influential outliers read counts on downstream ML analysis for sepsis biomarker discovery using elastic net regularised logistic regression, L1-reguarlised support vector machines and random forests. We demonstrate that applying a systematic objective strategy for removal of uninformative and potentially biasing biomarkers representing up to 60% of transcripts in different sample size datasets, including two illustrative neonatal sepsis cohorts, leads to substantial improvements in classification performance, higher stability of the resulting gene signatures, and better agreement with previously reported sepsis biomarkers. We also demonstrate that the performance uplift from gene filtering depends on the ML classifier chosen, with L1-regularlised support vector machines showing the greatest performance improvements with our experimental data.

Composition of early life leukocyte populations in preterm infants with and without late-onset sepsis.

BACKGROUND: Composition of leukocyte populations in the first month of life remains incompletely characterised, particularly in preterm infants who go on to develop late-onset sepsis (LOS). AIM: To characterise and compare leukocyte populations in preterm infants with and without LOS during the first month of life. STUDY DESIGN: Single-centre prospective observational cohort study. PARTICIPANTS: Infants born <30 weeks gestational age (GA). OUTCOME MEASURES: Peripheral blood samples were collected at 1, 7, 14, 21 and 28 days of life. Leukocyte populations were characterised using 5-fluorophore-6-marker flow cytometry. Absolute leukocyte counts and frequency of total CD45+ leukocytes of each population were adjusted for GA, birth weight z-scores, sex and total leukocyte count. RESULTS: Of 119 preterm infants enrolled, 43 (36%) had confirmed or clinical LOS, with a median onset at 13 days (range 6-26). Compared to infants without LOS, the adjusted counts and frequency of neutrophils, basophils and non-cytotoxic T lymphocytes were generally lower and immature granulocytes were higher over the first month of life in infants who developed LOS. Specific time point comparisons identified lower adjusted neutrophil counts on the first day of life in those infants who developed LOS more than a week later, compared to those without LOS, albeit levels were within the normal age-adjusted range. Non-cytotoxic T lymphocyte counts and/or frequencies were lower in infants following LOS on days 21 and 28 when compared to those who did not develop LOS. CONCLUSION: Changes in non-cytotoxic T lymphocytes occurred following LOS suggesting sepsis-induced immune suppression.

Plasma secretory phospholipase A2 as an early marker for late-onset sepsis in preterm infants-a pilot study.

Preterm infants are particularly susceptible to bacterial late-onset sepsis (LOS). Diagnosis by blood culture and inflammatory markers have sub-optimal sensitivity and specificity and prolonged reporting times. There is an urgent need for more rapid, accurate adjunctive diagnostics in LOS to improve management and minimise antibiotic exposure. We measured the diagnostic performance of secretory phospholipase A2 type IIA (sPLA2-IIA) in very preterm infants (<30 weeks gestational age) with suspected LOS. Plasma sPLA2-IIA levels were elevated in infants with LOS (n = 28) compared to those without LOS (n = 21; median 30,970 vs. 2534 pg/ml, p < 0.0001). The mean area under the curve was 0.884 (95% CI: 0.771, 0.977) with a sensitivity of 0.907 (95% CI: 0.667, 1.00) and specificity of 0.804 (95% CI: 0.600, 1.00). The positive and negative predictive values were 0.833 (95% CI: 0.664, 0.927) and 0.842 (95% CI: 0.624, 0.945), respectively. This pilot study suggests that sPLA2-IIA may have clinical utility for the early diagnosis of LOS in very preterm infants, potentially informing clinical management and antibiotic stewardship.

Impaired Cytokine Responses to Live Staphylococcus epidermidis in Preterm Infants Precede Gram-positive, Late-onset Sepsis.

BACKGROUND: Late-onset sepsis (LOS) with Staphylococcus epidermidis is common in preterm infants, but the immunological mechanisms underlying heightened susceptibility are poorly understood. Our aim is to characterize the ontogeny of cytokine responses to live S. epidermidis in preterm infants with and without subsequent Gram-positive LOS. METHODS: We conducted a prospective, observational cohort study of preterm infants (<30 weeks gestational age [GA]) with blood sampling on Days 1, 7, 14, 21, and 28 of life. Cytokine responses in peripheral whole blood stimulated with live S. epidermidis were analyzed by 11-plex immunoassay. RESULTS: Of 129 infants (mean GA, 26.2 weeks; mean birth weight, 887g), 23 (17.8%) had confirmed LOS with Gram-positive organisms and 15 (11.6%) had clinical sepsis, with median onsets at 13 and 15 days, respectively. Blood cytokine responses to an in vitro S. epidermidis challenge were similar between infected and uninfected infants on Day 1, but diverged thereafter. Infants with subsequent LOS displayed broadly reduced S. epidermidis-induced responses from Day 7 onwards, compared to those who did not develop LOS. This pattern was observed with chemokines (interleukin [IL]-8, monocyte chemotactic protein-1, and macrophage inflammatory protein-1α), pro-inflammatory cytokines (IL-1, IL-6, and tumor necrosis factor-α) and the regulatory cytokine IL-10. CONCLUSIONS: Cytokine responses to a live S. epidermidis challenge are impaired in infants with LOS and precede the onset of clinical illness. Quantifying pathogen-specific cytokine responses at Day 7 may identify those high-risk preterm infants at the greatest risk of LOS, and prospective replication is warranted.

The impact of cytokine levels in young South African children with and without HIV-associated acute lower respiratory infections.

Altered host immune responses are considered to play a key role in the pathogenesis of acute lower respiratory infections (ALRI). The existing literature on cytokine responses in ALRI is largely focussed on adults from developed countries and there are few reports describing the role of cytokines in childhood ALRI, particularly in African or human immunodeficiency virus (HIV)-infected populations. To measure systemic cytokine levels in blood plasma from young South African children with and without ALRI and with and without HIV to determine associations between cytokine responses and disease status and respiratory viral identification. Blood plasma samples were collected from 106 hospitalized ALRI cases and 54 non-ALRI controls less than 2 years of age. HIV status was determined. Blood plasma concentrations of 19 cytokines, 7 chemokines, and 4 growth factors (epidermal growth factor, fibroblast growth factor-basic, hepatocyte growth factor, and vascular endothelial) were measured using The Human Cytokine 30-Plex Panel. Common respiratory viruses were identified by PCR. Mean cytokine concentrations for G-CSF, interferon (IFN)-γ, interleukin (IL)-5, and MCP-1 were significantly higher in ALRI cases than in nonrespiratory controls. Within the ALRI cases, several cytokines were higher in children with a virus compared with children without a virus. Mean cytokine concentrations for IFN-α, IFN-γ, IL-4, IL-5, IL-13, tumour necrosis factor-α, and MIP-1α were significantly lower in HIV-infected cases than in HIV-uninfected cases, while IP-10 and monokine induced by interferon-γ were significantly higher in HIV-infected cases than in HIV-uninfected cases. Certain cytokines are likely to play an important role in the host immune response to ALRI. HIV-infected children have impaired inflammatory responses to respiratory infections compared with HIV-uninfected children.

Lactoferrin Expression Is Not Associated with Late-Onset Sepsis in Very Preterm Infants.

BACKGROUND: Preterm infants are at a high risk of developing late-onset sepsis (LOS). Lactoferrin is one of the most abundant endogenous antimicrobial proteins expressed in breast milk, stools, and blood, and a candidate for preventive intervention. Large clinical trials have recently investigated whether enteral supplementation with bovine lactoferrin reduces LOS. AIM: To characterize lactoferrin levels in preterm infants with and without LOS during the first month of life. METHODS: Very preterm and term infants were recruited and serial biosamples collected during the first month of life. Lactoferrin levels were determined by immunoassay in cord blood and peripheral blood on days 1, 7, 14, 21, and 28; in the stools on days 1 and 28; and in the mother's breast milk on days 7 and 21. Furthermore, we assessed the capacity of the peripheral blood to release lactoferrin in response to an in vitro challenge with live Staphylococcus epidermidis, lipopolysaccharide, and fibroblast-stimulating lipopeptide 1. RESULTS: Plasma lactoferrin levels were higher in cord blood and day 1 peripheral blood and declined during the first month of life. Plasma lactoferrin levels were similar in term infants and in preterm infants with (n = 32) and without LOS (n = 53). S. epidermidis-induced lactoferrin levels were lower following the sepsis episode. CONCLUSIONS: Endogenous lactoferrin expression in preterm infants does not appear to affect their risk of developing LOS. These findings are in line with the lack of benefit recently observed in large trials of enteral supplementation with bovine lactoferrin to prevent LOS.

Whole blood transcriptional responses of very preterm infants during late-onset sepsis.

BACKGROUND: Host immune responses during late-onset sepsis (LOS) in very preterm infants are poorly characterised due to a complex and dynamic pathophysiology and challenges in working with small available blood volumes. We present here an unbiased transcriptomic analysis of whole peripheral blood from very preterm infants at the time of LOS. METHODS: RNA-Seq was performed on peripheral blood samples (6-29 days postnatal age) taken at the time of suspected LOS from very preterm infants <30 weeks gestational age. Infants were classified based on blood culture positivity and elevated C-reactive protein concentrations as having confirmed LOS (n = 5), possible LOS (n = 4) or no LOS (n = 9). Bioinformatics and statistical analyses performed included pathway over-representation and protein-protein interaction network analyses. Plasma cytokine immunoassays were performed to validate differentially expressed cytokine pathways. RESULTS: The blood leukocyte transcriptional responses of infants with confirmed LOS differed significantly from infants without LOS (1,317 differentially expressed genes). However, infants with possible LOS could not be distinguished from infants with no LOS or confirmed LOS. Transcriptional alterations associated with LOS included genes involved in pathogen recognition (mainly TLR pathways), cytokine signalling (both pro-inflammatory and inhibitory responses), immune and haematological regulation (including cell death pathways), and metabolism (altered cholesterol biosynthesis). At the transcriptional-level cytokine responses during LOS were characterised by over-representation of IFN-α/ß, IFN-γ, IL-1 and IL-6 signalling pathways and up-regulation of genes for inflammatory responses. Infants with confirmed LOS had significantly higher levels of IL-1α and IL-6 in their plasma. CONCLUSIONS: Blood responses in very preterm infants with LOS are characterised by altered host immune responses that appear to reflect unbalanced immuno-metabolic homeostasis.

Plasma cytokine profiles in very preterm infants with late-onset sepsis.

INTRODUCTION: Deficiencies in innate immune responses may contribute to the increased susceptibility to infection in preterm infants. In vivo cytokine profiles in response to sepsis in very preterm infants are not fully understood. AIMS: To characterise plasma pro- and anti-inflammatory cytokine concentrations and pre-defined ratios in very preterm infants with late-onset sepsis (LOS). METHODS: In this observational study, peripheral blood samples were collected at the time of evaluation for suspected LOS from 31 preterm infants (<30 weeks gestational age). Plasma cytokine concentrations were determined by 12-plex immunoassay. RESULTS: IL-10, IFN-γ, IL-12p70, IP-10, IL-6 and CCL2 were elevated in the majority infants with LOS (n = 12) compared to those without LOS (n = 19). There was no difference in TNF-α, IL-1ß, IL-17AF, IL-8 and IL-15 concentrations between groups. IL-10/TNF-α ratios were increased, while CCL2/IL-10 and IL-12p70/IL-10 ratios were decreased in infants with LOS compared to those without. CONCLUSION: Very preterm infants have a marked innate inflammatory response at the time of LOS. The increase in IL-10/TNF-α ratio may indicate early immune hypo-responsiveness. Longitudinal studies with a larger number of participants are required to understand immune responses and clinical outcomes following LOS in preterm infants.

Cyclic AMP in human preterm infant blood is associated with increased TLR-mediated production of acute-phase and anti-inflammatory cytokines in vitro.

BACKGROUND: Preterm infants are at high risk of infection and have distinct pathogen recognition responses. Suggested mechanisms include soluble mediators that enhance cellular levels of cAMP. The aim of this study was to assess the relationship between blood cAMP concentrations and TLR-mediated cytokine production in infants during the first month of life. METHODS: Cord and serial peripheral blood samples (days of life 1-28) were obtained from a cohort of very preterm (<30 weeks' gestational age) and term human infants. Whole-blood concentrations of cAMP and FSL-1 and LPS in vitro stimulated cytokine concentrations were measured by ELISA and multiplex bead assay. RESULTS: cAMP concentrations were higher in cord than in peripheral blood, higher in cord blood of female preterm infants, and lower at Days 1 and 7 in infants exposed to chorioamnionitis, even after adjusting for leukocyte counts. TLR2 and TLR4-mediated TNF-α, IL-1ß, IL-6, IL-12p70, and IL-10 production in vitro increased over the first month of life in preterm infants and were positively correlated with leukocyte-adjusted cAMP levels and reduced by exposure to chorioamnionitis. CONCLUSIONS: The ontogeny of blood cAMP concentrations and associations with chorioamnionitis and TLR-mediated production of cytokines suggest that this secondary messenger helps shape distinct neonatal pathogen responses in early life.

High concentrations of middle ear antimicrobial peptides and proteins and proinflammatory cytokines are associated with detection of middle ear pathogens in children with recurrent acute otitis media.

Recurrent and chronic otitis media (OM) are often refractory to antibiotics due to bacterial persistence in biofilm within the middle ear. In vitro and in vivo studies have demonstrated that antimicrobial proteins and peptides (AMPs) are bactericidal against otopathogens, indicating potential therapeutic value for recalcitrant OM. We measured concentrations of 6 AMPs and 14 cytokines in middle ear effusion (MEE) from 67 children undergoing ventilation tube insertion for recurrent acute OM. Sixty one percent of children had bacterial otopathogens detected in their MEE, 39% by PCR and 22% by PCR and culture. Groups were defined as: PCR-negative/culture-negative (absence of bacterial otopathogen), n = 26; PCR-positive/culture-negative (presence of nonculturable bacterial otopathogen), n = 26; PCR-positive/culture-positive (presence of culturable bacterial otopathogen), n = 15. Age, antibiotic usage, day-care attendance, presence of respiratory viruses in MEE and number of AOM episodes were similar between groups. AMP and cytokine concentrations were higher in children with bacterial otopathogens in their MEE compared to those with no bacterial otopathogens. Median concentrations of AMPs (except HBD2) were 3 to 56-fold higher in MEE from children with bacterial otopathogens detected in their MEE (P ≤ 0.01). Similarly, median cytokine concentrations (except TGFß) were >16-fold higher in MEE with bacterial otopathogens detected (P ≤ 0.001). This is the first study to measure AMPs in MEE and together with the cytokine data, results suggest that elevated AMPs and cytokines in MEE are a marker of inflammation and bacterial persistence. AMPs may play an important role in OM pathogenesis.

Effects of maturation and size on population pharmacokinetics of pentoxifylline and its metabolites in very preterm infants with suspected late-onset sepsis or necrotizing enterocolitis: a pilot study incorporating clinical outcomes.

AIMS: Infection-induced inflammation is associated with adverse long-term outcomes in preterm infants. Pentoxifylline (PTX) is a candidate for adjunct immunomodulatory therapy in preterm infants with late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), but pharmacokinetic data in this population are extremely limited. This study aims to characterize the pharmacokinetic properties of intravenous PTX and its metabolites in preterm infants. METHOD: An open label pilot clinical study of intravenous PTX as an adjunct therapy in preterm infants (gestation <32 weeks) with suspected LOS or NEC was undertaken. PTX was infused for 12 h for two days (60 mg kg-1 per 12 h), and in infants with confirmed diagnosis of LOS or NEC, for 6 h for another 4 days (30 mg kg-1 per 6 h). Plasma concentrations of PTX and its principal metabolites from collected blood samples were measured using a validated LCMS assay. NONMEM was used to analyse the data using population pharmacokinetic modelling. RESULTS: The preterm infants (n = 26) had a median (range) gestation of 24.8 weeks (23.3-30.4) and birthweight of 689 g (370-1285). PTX was well tolerated and without treatment-limiting adverse effects. Changes in size (weight) and maturation were successfully modelled for PTX and metabolites. After allometric scaling, clearance increased with postmenstrual age, increasing by approximately 30% per week for PTX and M1 (lisofylline) and simulations of current dosing demonstrated a six-fold difference in exposure between 24 and 35 weeks postmenstrual age. CONCLUSIONS: The developed model can be used to explore dosing strategies based on size and maturation for preterm infants.

Sepsis-Induced Immunosuppression in Neonates.

Neonates, especially those born preterm, are at increased risk of sepsis and adverse long-term effects associated with infection-related inflammation. Distinct neonatal immune responses and dysregulated inflammation are central to this unique susceptibility. The traditional separation of sepsis into an initial hyper-inflammatory response followed by hypo-inflammation is continually under review with new developments in this area of research. There is evidence to support the association of mortality in the early acute phase of sepsis with an overwhelming hyper-inflammatory immune response. Emerging evidence from adults suggests that hypo- and hyper-inflammation can occur during any phase of sepsis and that sepsis-immunosuppression is associated with increased mortality, morbidity, and risk to subsequent infection. In adults, sepsis-induced immunosuppression (SII) is characterised by alterations of innate and adaptive immune responses, including, but not limited to, a prominent bias toward anti-inflammatory cytokine secretion, diminished antigen presentation to T cells, and reduced activation and proliferation of T cells. It is unclear if sepsis-immunosuppression also plays a role in the adverse outcomes associated with neonatal sepsis. This review will focus on exploring if key characteristics associated with SII in adults are observed in neonates with sepsis.

Correction: Human alkaline phosphatase dephosphorylates microbial products and is elevated in preterm neonates with a history of late-onset sepsis.

[This corrects the article DOI: 10.1371/journal.pone.0175936.].

Topical Coconut Oil in Very Preterm Infants: An Open-Label Randomised Controlled Trial.

BACKGROUND: The immature fragile skin of preterm infants represents an inadequate protective barrier. The emollient and anti-infective properties of coconut oil make it a potentially beneficial topical agent for this population. OBJECTIVES: Our aim was to evaluate feasibility, safety, and the effects of topical coconut oil on skin condition in very preterm infants. METHODS: An open-label randomised controlled trial in preterm infants <30 weeks' gestation was conducted. Enrolled infants were randomised to receive either routine care or topical coconut oil (5 mL/kg) twice daily for 21 days, starting within 24 h of birth. The neonatal skin condition was the primary outcome, and was assessed using the Neonatal Skin Condition Score (NSCS) on days 1, 7, 14, and 21. The number of coconut oil applications was recorded to assess clinical feasibility and all enrolled infants were monitored for adverse effects of topical coconut application, such as skin irritation. RESULTS: A total of 72 infants born <30 weeks' gestation were enrolled (36 infants per arm), with comparable demographic characteristics. Topical application of coconut oil was feasible and without adverse effects. The NSCS was maintained in the coconut oil group throughout the intervention period, but deteriorated from a median (IQR) of 3 (3-4) on day 1 to 4 (4-4) on day 21 in the control group (p = 0.01). There were no differences in common neonatal outcomes, including sepsis, necrotising enterocolitis, retinopathy of prematurity, chronic lung disease, and mortality. CONCLUSIONS: Topical coconut oil maintained a better skin condition in very preterm infants without adverse effects. This simple, safe, and affordable intervention warrants further investigation.

Identification of generic and pathogen-specific cord blood monocyte transcriptomes reveals a largely conserved response in preterm and term newborn infants.

Escherichia coli and Staphylococcus epidermidis are predominant causes of neonatal sepsis, particularly affecting preterm infants. Susceptibility to infection has been attributed to "immature" innate monocyte defences, but no studies have assessed global transcriptional responses of neonatal monocytes to these pathogens. Here, we aimed to identify and characterise the neonatal monocyte transcriptional responses to E. coli and S. epidermidis and the role of common modifiers such as gestational age (GA) and exposure to chorioamnionitis (a common complication of preterm birth) to better understand early life innate immune responses. RNA-sequencing was performed on purified cord blood monocytes from very preterm (< 32 weeks GA) and term infants (37-40 weeks GA) following standardised challenge with live S. epidermidis or E. coli. The major transcriptional changes induced by either pathogen were highly conserved between infant groups and stimuli, highlighting a common extant neonatal monocyte response to infection, largely mediated by TLR/NF-κB/TREM-1 signalling. In addition, we observed an activated interferon-centred immune response specific to stimulation with E. coli in both preterm and term infants. These data provide novel insights into the functionality of neonatal monocytes at birth and highlight potential pathways that could be targeted to reduce the harmful effects of bacterial-induced inflammation in sepsis. E. coli and S. epidermidis elicit common transcriptional changes in cord monocytes. The common transcriptional response is mediated by TLR/NF-κB/TREM-1 signalling. IFN genes are differentially regulated by E. coli and S. epidermidis in monocytes. These responses are largely unaffected by GA or exposure to chorioamnionitis. KEY MESSAGES: E. coli and S. epidermidis elicit common transcriptional changes in cord monocytes. The common transcriptional response is mediated by TLR/NF-κB/TREM-1 signalling. IFN-genes are differentially regulated by E. coli and S. epidermidis in monocytes. These responses are largely unaffected by GA or exposure to chorioamnionitis.

Simultaneous determination of pentoxifylline, metabolites M1 (lisofylline), M4 and M5, and caffeine in plasma and dried blood spots for pharmacokinetic studies in preterm infants and neonates.

Advances in bioanalytical methods are facilitating micro-volume and dried blood spot (DBS) analysis of drugs in biological matrices for pharmacokinetic studies in children and neonates. We sought to develop a UPLC-MS/MS assay for simultaneous measurement of caffeine, pentoxifylline (PTX) and three metabolites of PTX in both plasma and DBS. Caffeine, PTX, the metabolites M1 (lisofylline), M4 and M5, and the internal standards (caffeine-d9 and PTX-d6) were separated using a Waters Aquity T3 UPLC C18 column and gradient mobile phase (water-methanol-formic acid). Retention times for caffeine, M5, M4, PTX and M1 were 1.6, 1.7, 1.9, 2.0 and 2.1min, respectively, with a run time of 5min. The precision (≤10%) and accuracy (≤15%) across the concentration range 0.1-50mg/L for caffeine, PTX and the three metabolites in plasma and DBS were within accepted limits, as were the limits of quantification (100µg/L for caffeine and 10µg/L for PTX, M1, M4 and M5). Caffeine, PTX and the metabolites were stable in DBS for >34days at room and refrigerated temperatures. Plasma and DBS samples were obtained from 24 preterm infants recruited into a clinical pharmacokinetic study of PTX. Paired analysis indicated that DBS concentrations were 9% lower than concurrent plasma concentrations for caffeine, 7% lower for PTX (consistent with the blood:plasma ratio) and 13% lower for M1 (lisofylline). The validated UPLC-MS/MS method is suitable for micro-volume plasma and DBS analysis of caffeine, PTX and its metabolites for pharmacokinetic studies in paediatric patients.

Human alkaline phosphatase dephosphorylates microbial products and is elevated in preterm neonates with a history of late-onset sepsis.

BACKGROUND: A host defense function for Alkaline phosphatases (ALPs) is suggested by the contribution of intestinal ALP to detoxifying bacterial lipopolysaccharide (endotoxin) in animal models in vivo and the elevation of ALP activity following treatment of human cells with inflammatory stimuli in vitro. However the activity of ALP in human plasma (primarily tissue-nonspecific ALP; TNAP) on lipopolysaccharide and other microbial products has not been assessed, nor has its expression been studied in preterm newborns, a vulnerable population at high risk of sepsis. In this context, the aim of our study was to characterize the activity of TNAP on Toll-like receptor (TLR) agonists and assess the concentrations of plasma ALP during late-onset sepsis in preterm newborns. METHODS: Recombinant human TNAP was incubated with microbial products and phosphate release was measured by malachite green assay. Plasma ALP activity was measured serially in a cohort of preterm (N = 129) infants at high risk of late-onset sepsis (LOS). RESULTS: TNAP dephosphorylates poly-inosine:cytosine (Toll-like receptor (TLR) 3 agonist) and LPS from Klebsiella pneumoniae and Salmonella minnesota (TLR4 agonists). Plasma ALP significantly increased postnatally over the first 4 weeks of life in preterm and term newborns. Bacteremic LOS in preterm infants (gestational age ≤ 30 weeks) was associated with significantly elevated plasma ALP at 4 weeks postnatal age. CONCLUSIONS: TNAP, the main circulating isozyme of ALP, de-phosphorylates TLR agonists, demonstrates a post-natal age dependent increase in preterm and term plasma across the first 4 weeks of life, and is elevated in association with preterm LOS.