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Objective: We compared mortality and morbidity of inborn versus outborn very preterm infants <32 weeks' gestation in Western Australia (WA) between 2005 and 2018., Design: Retrospective cohort study., Patients: Infants <32 weeks' gestation who were born in WA., Main Outcome Measures: Mortality was assessed as death before discharge home from the tertiary neonatal intensive care unit. Short-term morbidities included combined brain injury (intracranial haemorrhage grade ≥3 and cystic periventricular leukomalacia) and other major neonatal outcomes. Developmental assessments at age 2, 3 and 5 years were evaluated. We performed multivariable logistic regression analysis of outborn status on outcomes, controlling for gestational age, birth weight z-score, sex and multiple birth., Results: A total of 4974 infants were born in WA between 22 and 32 weeks' gestation between 2005 and 2018 of which 4237 (89.6%) were inborn and 443 (10.4%) were outborn. Overall mortality to discharge was higher in outborn infants (20.5% (91/443) vs 7.4% (314/4237); adjusted OR (aOR) 2.44, 95% CI 1.60 to 3.70, p<0.001). Outborn infants had higher rates of combined brain injury than those inborn (10.7% (41/384) vs 6.0% (246/4115); aOR 1.98, 95% CI 1.37 to 2.86), p<0.001). No difference in up to 5-year developmental measures was detected. Follow-up data were available for 65% of outborn and 79% of inborn infants., Conclusions: Outborn preterm infants <32 weeks in WA had increased odds of mortality and combined brain injury than those inborn. Developmental outcomes up to 5 years were similar between groups. Loss to follow-up may have impacted the long-term comparison., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: The authors have declared that no competing interests exist.

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-1β, 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.

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-1β, 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., Competing Interests: The authors have declared that no competing interests exist.

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.

Aim: To characterise the secreted and inducible antimicrobial protein and peptide (APP) levels in a prospective cohort of preterm infants (<30 weeks gestational age) with or without Bifidobacterium breve M16V supplementation during the first month of life., Methods: We analysed serial biosamples of infants who did (n = 13) or did not receive (n = 62) B. breve (3 × 10 9 cfu/day). Peripheral blood was obtained on days 1, 14 and 28, and infant stool prior to commencement of probiotic supplementation and on day 21. Levels of APP (bactericidal/permeability inducing protein (BPI), beta defensins 1 and 2, lactoferrin, human cathelicidin, secretory phospholipase A2) in plasma and stool were determined. Further, we characterised induced APP levels in whole blood cultured with live S. epidermidis or with agonists of Toll-like receptors 2/6 and 4., Results: Stool, plasma and stimulated blood APP levels changed significantly during the first month of life. Supplementation with B. breve did not affect basal or stimulated APP levels except for a transient increase in inducible BPI., Conclusion: Supplementation with B. breve does not appear to act via modulation of systemic or enteric APP expression in preterm infants although small effects cannot be excluded. Further work with other probiotic preparations is warranted., (©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

The extreme vulnerability of preterm infants to invasive microbial infections has been attributed to "immature" innate immune defenses. Monocytes are important innate immune sentinel cells critical in the defense against infection in blood. They achieve this via diverse mechanisms that include pathogen recognition receptor- and inflammasome-mediated detection of microbes, migration into infected tissues, and differentiation into Mϕs and dendritic cells, initiation of the inflammatory cascade by free radicals and cytokine/chemokine production, pathogen clearance by phagocytosis and intracellular killing, and the removal of apoptotic cells. Relatively little is known about these cells in preterm infants, especially about how their phenotype adapts to changes in the microbial environment during the immediate postnatal period. Overall, preterm monocytes exhibit attenuated proinflammatory cytokine responses following stimulation by whole bacterial or specific microbial components in vitro. These attenuated cytokine responses cannot be explained by a lack of intracellular signaling events downstream of pattern recognition receptors. This hyporesponsiveness also contrasts with mature, term-like phagocytosis capabilities detectable even in the most premature infant. Finally, human data on the effects of fetal chorioamnionitis on monocyte biology are incomplete and inconsistent. In this review, we present an integrated view of human studies focused on monocyte functions in preterm infants. We discuss how a developmental immaturity of these cells may contribute to preterm infants' susceptibility to infections., (© Society for Leukocyte Biology.)

Background: Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood., Methods: Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28-31 wk), and moderately preterm (32-36 wk), as well as term (37-41 wk) infants were recruited. Colostrum (d2-5), transitional (d8-12) and mature milk (d26-30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry., Results: The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed., Conclusions: Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk.

Objective: We investigated the levels and antimicrobial activity of antimicrobial proteins and peptides (AMPs) in breast milk consumed by preterm infants, and whether deficiencies of these factors were associated with late-onset neonatal sepsis (LOS), a bacterial infection that frequently occurs in preterm infants in the neonatal period., Study Design: Breast milk from mothers of preterm infants (≤ 32 weeks gestation) was collected on days 7 (n = 88) and 21 (n = 77) postpartum. Concentrations of lactoferrin, LL-37, beta-defensins 1 and 2, and alpha-defensin 5 were measured by enzyme-linked immunosorbent assay. The antimicrobial activity of breast milk samples against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae was compared to the activity of infant formula, alone or supplemented with physiological levels of AMPs. Samples of breast milk fed to infants with and without subsequent LOS were compared for levels of AMPs and inhibition of bacterial growth., Results: Levels of most AMPs and antibacterial activity in preterm breast milk were higher at day 7 than at day 21. Lactoferrin was the only AMP that limited pathogen growth >50% when added to formula at a concentration equivalent to that present in breast milk. Levels of AMPs were similar in the breast milk fed to infants with and without LOS, however, infants who developed LOS consumed significantly less breast milk and lower doses of milk AMPs than those who were free from LOS., Conclusions: The concentrations of lactoferrin and defensins in preterm breast milk have antimicrobial activity against common neonatal pathogens.

Preterm birth and infectious diseases are the most common causes of neonatal and early childhood deaths worldwide. The rates of preterm birth have increased over recent decades and account for 11% of all births worldwide. Preterm infants are at significant risk of severe infection in early life and throughout childhood. Bacteraemia, inflammation, or both during the neonatal period in preterm infants is associated with adverse outcomes, including death, chronic lung disease, and neurodevelopmental impairment. Recent studies suggest that bacteraemia could trigger cerebral injury even without penetration of viable bacteria into the CNS. Here we review available evidence that supports the concept of a strong association between bacteraemia, inflammation, and cerebral injury in preterm infants, with an emphasis on the underlying biological mechanisms, clinical correlates, and translational opportunities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aim: To evaluate mononuclear cell expression and function of the cytosolic nucleotide-binding oligomerization domain-containing receptors, NOD1 and NOD2, in very preterm and full-term infants., Methods: NOD1 and NOD2 gene and protein expression in very preterm infants, term infants and healthy adult, cord and peripheral blood mononuclear cells (C/PBMC) were quantified using qPCR and flow cytometry. Cytokine responses of purified infant and adult monocytes to NOD1- and NOD2-specific agonists were assessed using a multiplex immunoassay (Bioplex)., Results: NOD1 and NOD2 were expressed by a range of infant and adult mononuclear cell types, including T- and B cells, with highest expression in classical (CD14(++) CD16(-) ) and intermediate (CD14(++) CD16(+) ) monocytes. NOD1 and NOD2 expression levels by monocytes from very preterm infant were similar to those in term infants or adults. Monocyte production of TNFα, IL-6 and IL-1β induced by activation of NOD1 and NOD2 was similar between very preterm infants, term infants and adults., Conclusion: Monocyte expression and function of NOD1 and NOD2 in very preterm infants are intact and comparable/equivalent to term infants and adults. Functional deficiencies in monocyte NOD signalling pathways are unlikely to contribute to the increased susceptibility to bacterial sepsis in preterm infants., (©2014 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

Background: Deficiencies in phagocytosis may contribute to the increased susceptibility of infants to early life infections. Data on phagocytosis of the major neonatal pathogens Staphylococcus epidermidis (SE), Staphylococcus aureus (SA), and Escherichia coli (EC) by preterm infant leukocytes are inconsistent., Methods: Cord and <24-h peripheral blood were collected from very preterm (<30.1 wks gestational age (GA)) and term (37-42 wks GA) infants. Monocyte and neutrophil phagocytosis of pHrodo-labeled SE, SA, and EC were analyzed using a small-volume flow cytometry assay, with simultaneous characterization of surface activation marker expression., Results: Preterm infants had lower proportions of monocytes and neutrophils capable of phagocytosis than term infants, but preterm infant phagocytes had higher phagocytic capacity. Phagocytosis was strongly correlated between cord and <24-h peripheral blood. Supplementation with exogenous complement significantly increased phagocytosis of EC but not of SE or SA. Monocyte human leukocyte antigen (HLA)-DR expression was lower in preterm infants but did not correlate with phagocytosis., Conclusion: There is no defect in phagocytosis by monocytes and neutrophils from preterm compared with term infants, although preterm infants possess fewer phagocytes, possibly contributing to susceptibility to bacterial infection. Further investigation into the development of postnatal phagocytic competence is warranted.

Group B streptococcus (GBS) is an important cause of early- and late-onset sepsis in the newborn. Preterm infants have markedly increased susceptibility and worse outcomes, but their immunological responses to GBS are poorly defined. We compared mononuclear cell and whole-blood cytokine responses to heat-killed GBS (HKGBS) of preterm infants (gestational age [GA], 26 to 33 weeks), term infants, and healthy adults. We investigated the kinetics and cell source of induced cytokines and quantified HKGBS phagocytosis. HKGBS-induced tumor necrosis factor (TNF) and interleukin 6 (IL-6) secretion was significantly impaired in preterm infants compared to that in term infants and adults. These cytokines were predominantly monocytic in origin, and production was intrinsically linked to HKGBS phagocytosis. Very preterm infants (GA, <30 weeks) had fewer cytokine-producing monocytes, but nonopsonic phagocytosis ability was comparable to that for term infants and adults. Exogenous complement supplementation increased phagocytosis in all groups, as well as the proportion of preterm monocytes producing IL-6, but for very preterm infants, responses were still deficient. Similar defective preterm monocyte responses were observed in fresh whole cord blood stimulated with live GBS. Lymphocyte-associated cytokines were significantly deficient for both preterm and term infants compared to levels for adults. These findings indicate that a subset of preterm monocytes do not respond to GBS, a defect compounded by generalized weaker lymphocyte responses in newborns. Together these deficient responses may increase the susceptibility of preterm infants to GBS infection.

Neonates, particularly those born prematurely, are exquisitely vulnerable to life-threatening infections. This increased susceptibility to infection is maintained into childhood. Despite the considerable human and economic cost of infection-related neonatal morbidity and mortality, the mechanisms underlying this heightened susceptibility are only partly understood. It is increasingly recognised that innate immune responses are key to the protection against infection early in life, and emerging data suggest that such responses are deficient in the newborn and especially in preterm infants. Here we review the current understanding of the maturation of the innate immune response in human neonates highlighting the clinical relevance and possible avenues for therapeutic intervention.