Neonatal Neutrophils Research Articles

Neonatal neutrophils exhibit reduced NLRP3 inflammasome activation

Abstract Newborns are at high risk to develop sepsis. This is linked to innate immune responses at birth which are not completely adapted to postnatal life. Neutrophils are key players of innate immunity and exhibit a marked ontogenetic regulation of their functionality. Here, we studied the NLRP3 inflammasome in neonatal neutrophils and found lower baseline expression of NLRP3, pro-caspase-1 and the K+-channel KV1.3 compared to adult neutrophils. Following stimulation with LPS/Nigericin, ASC oligomerization, caspase-1 activation and IL-1β release were significantly reduced in neonatal compared to adult neutrophils. Similarly, stimulation of neonatal neutrophils with E-selectin led to reduced NLRP3 inflammasome activation accompanied by diminished release of the alarmin S100A8/A9. Taken together, our results strongly indicate diminished NLRP3 inflammasome activation in neonatal neutrophils leading to a significant reduction of released IL-1β and S100A8/A9. These findings identify reduced neutrophil NLRP3 inflammasome activation as critical component contributing to the inherent susceptibility to infections in neonates.

Protocols for circulating neutrophil depletion in neonatal C57Bl/6 mice.

Murine neonatal neutrophil depletion strategies have problems with achieving deep circulating neutrophil clearance and accurate residual neutrophil fraction detection. Boivin et al. demonstrated an isotype switch method of achieving profound neutrophil clearance using a combination of anti-Ly6G and anti-rat κ Ig light chain antibodies in adult C57Bl/6 mice, proven by extra- and intracellular Ly6G detection by flow cytometry. We adapted this technique to neonatal mice testing four neutrophil depletion strategies. Four protocols were tested: P3 Ly6G and P1-3 Ly6G (anti-Ly6G on postnatal days (P) 3 and 1-3 respectively), and P3 Dual and P1-3 Dual (anti-Ly6G and anti-rat κ Ig light chain on P3 and P1-3 respectively). Intracellular and extracellular Ly6G presence was detected using flow cytometry. Isotype control antibodies were used as controls. P1-3 Dual achieved significantly better neutrophil depletion than the P1-3 Ly6G or P3 Ly6G protocols (97% vs 74% and 97% vs 50%, respectively). The P3 Dual protocol alone was enough to achieve significantly better neutrophil clearance (93%) than any of the Ly6G alone protocols. The Ly6G alone protocols led to near-total elimination of extracellular Ly6G, but there was a significant presence of intracellular Ly6G in the CD45+ cell population, which evaded detection by extracellular Ly6G antibody-based detection methods. Anti-Ly6G antibody alone is ineffective in producing effective circulating neutrophil clearance in neonatal mice. Dual antibody-based neutrophil depletion strategies achieve>90% clearance in P4 mice. A single day of dual antibody treatment achieves 93% neutrophil depletion in neonatal mice and is an alternative to daily injections. Dual antibody-based neutrophil depletion effectively induces circulating neutrophil clearance in neonatal mice.

The neonatal liver hosts a spontaneously occurring neutrophil population, exhibiting distinct spatial and functional characteristics from adults.

The elusive nature of the liver immune system in newborns remains an important challenge, casting a shadow over our understanding of how to effectively treat and prevent diseases in children. Therefore, deeper exploration into the intricacies of neonatal immunology might be crucial for improved pediatric healthcare. Using liver intravital microscopy, we unveiled a significant population of granulocytes in the hepatic parenchyma of fetuses and newborns. Utilizing high-dimensional immunophenotyping, we showed dynamic alterations predominantly in granulocytes during neonatal development. Liver intravital microscopy from birth through adulthood captures real-time dynamics, showing a substantial presence of Ly6G + cells that persisted significantly up to 2 weeks of age. Using CyTOF, we characterized neonatal Ly6G + cells as neutrophils, confirmed by morphology and immunohistochemistry. Surprisingly, the embryonic liver hosts a distinct population of neutrophils established as early as the second gestational week, challenging conventional notions about their origin. Additionally, we observed that embryonic neutrophils occupy preferentially the extravascular space, indicating their early establishment within the liver. Hepatic neutrophils in embryos and neonates form unique cell clusters, persisting during the initial days of life, while reduced migratory capabilities in neonates are observed, potentially compensating with increased reactive oxygen species (ROS) release in response to stimuli. Finally, in vivo imaging of acute neutrophil behavior in a newborn mouse, subjected to focal liver necrosis, unveils that neonatal neutrophils exhibit a reduced migratory response. The study provides unprecedented insights into the intricate interplay of neutrophils within the liver, shedding light on their functional and dynamic characteristics during development.

Maternal Hypertension and Early-Onset Neonatal Neutropenia

Objective: Maternal hypertension is considered a risk factor for early neonatal neutropenia. We sought to explore this relationship. Study Design: This retrospective cohort study compared initial neutrophil counts in infants born to mothers with preeclampsia with severe features (PSF) and infants born to normotensive mothers using Negative Binomial Regression (NBR) and logistic regression models. Results: Maternal hypertension negatively affected the early neonatal neutrophil count (adjusted NRB coefficient 0.4 [0.2, 0.6], p < 0.0001) but did not increase the risk of neutropenia (OR 2.07 [0.97, 4.41], p = 0.06). The initial neutrophil count and neutropenia risk were not different between PSF subgroups. Gestational age had the greatest impact on neutropenia risk (OR 0.72 [0.64, 0.81], p < 0.0001). Almost all neutropenia resolved within 48 h. Conclusion: Maternal hypertension negatively affects the early neonatal neutrophil count while not increasing the risk of neonatal neutropenia.

RAC2 mutations and immune deficiency – functional spectrum of an international cohort

Mutations in RAC2 were first identified in neonates carrying RAC2 dominant negative p.D57N, who presented with severe neutrophil defects and T-cell lymphopenia. Subsequently, dominant-activating mutations were reported in patients with combined immunodeficiency/immunodysregulation (CIID) or severe combined immunodeficiency (SCID). We assembled an international cohort of 48 published and unpublished patients with RAC2 mutations to characterize the spectrum of disease.Clinical and immunologic phenotypes of 48 germline mutant RAC2 patients were obtained from referring physicians and literature reports. All patients had significant T lymphopenia with several patients detected by newborn screening, most patients exhibited B and NK lymphopenia as well. Respiratory infections were common, affecting 90% of patients while 56% had significant viral infections and 15% had malignancies. Heterologous RAC2 expression assays including superoxide production, PAK1 binding, and AKT activation were used to characterize RAC2 variant effects on downstream signaling. Protein stability was determined by Western blot. Patient presentation varied by mutation: five patients presented as SCID with reticular dysgenesis, five as neonatal neutrophil deficiency (D57N), and 38 as CIID. Patients with dominant, active RAC2 variants with high protein stability (Q61R, Q61K) presented in the first days of life with SCID and absent lymphocytes. Activating variants with lower stability (C18W, G30R, E62K, N92T, R174) presented as CIID with lymphopenia, detectable by newborn screening but presenting later with recurrent sinopulmonary and viral infections. A third class of mutations with unstable transcript or protein were phenotypic only in homozygosity (W56*, R68W). Consistent with the role of RAC2 in the NADPH oxidase complex, patients with mutations causing reduced superoxide production exhibited bacterial infections. Severity of phenotype and initial clinical presentation were correlated with protein stability such that high stability led to neonatal presentation whereas dominant mutations with decreased stability had later clinical onset.Clinical presentation of RAC2 mutation-bearing patients is determined by a combination of protein activity and stability. Heterologous expression together with RAC2 functional analysis is useful for deciphering the molecular basis of the clinical phenotype related to these immunodeficiencies.

Depletion of Ly6G-Expressing Neutrophilic Cells Leads to Altered Peripheral T-Cell Homeostasis and Thymic Development in Neonatal Mice.

Newborns and especially preterm infants are much more susceptible to infections than adults. Due to immature adaptive immunity, especially innate immune cells play an important role in a newborn's infection defense. Neonatal neutrophils exhibit profound differences in their functionality compared to neutrophils of adults. In particular, neonates possess a relevant population of suppressive neutrophils, which not only inhibit but also specifically modulate the function of T-cells. In this study, we investigated whether neonatal neutrophils are already involved in T-cell development in the thymus. For this purpose, we used a newly developed model of antibody-mediated immune cell depletion in which we administered a depleting antibody to pregnant and then lactating dams. Using this method, we were able to sufficiently deplete Ly6G-positive neutrophils in offspring. We demonstrated that the depletion of neutrophils in newborn mice resulted in altered peripheral T-cell homeostasis with a decreased CD4+/CD8+ T-cell ratio and decreased expression of CD62L. Neutrophil depletion even affected T-cell development in the thymus, with increased double positive thymocytes and a decreased CD4+/CD8+ single positive thymocyte ratio. Altogether, we demonstrated a previously unknown mechanism mediating neutrophils' immunomodulatory effects in newborns.

Age-dependent differences in efferocytosis determine the outcome of opsonophagocytic protection from invasive pathogens

In early life, susceptibility to invasive infection skews toward a small subset of microbes, whereas other pathogens associated with diseases later in life, including Streptococcus pneumoniae (Spn), are uncommon among neonates. To delineate mechanisms behind age-dependent susceptibility, we compared age-specific mouse models of invasive Spn infection. We show enhanced CD11b-dependent opsonophagocytosis by neonatal neutrophils improved protection against Spn during early life. The augmented function of neonatal neutrophils was mediated by higher CD11b surface expression at the population level due to dampened efferocytosis, which also resulted in more CD11bhi "aged" neutrophils in peripheral blood. Dampened efferocytosis during early life could be attributed to the lack of CD169+ macrophages in neonates and reduced systemic expressions of multiple efferocytic mediators, including MerTK. On experimentally impairing efferocytosis later in life, CD11bhi neutrophils increased and protection against Spn improved. Our findings reveal how age-dependent differences in efferocytosis determine infection outcome through the modulation of CD11b-driven opsonophagocytosis and immunity.

Olfactomedin-4-Positive Neutrophils in Neonates: Link to Systemic Inflammation and Bronchopulmonary Dysplasia

Introduction: Little is known about the interplay between neutrophil heterogeneity in neonates in health and disease states. Olfactomedin-4 (OLFM4) marks a subset of neutrophils that have been described in adults and pediatric patients but not neonates, and this subset is thought to play a role in modulating the host inflammatory response. Methods: This is a prospective cohort of neonates who were born between June 2020 and December 2021 at the University of Cincinnati Medical Center NICU. Olfactomedin-4-positive (OLFM4+) neutrophils were identified in the peripheral blood using flow cytometry. Results: OLFM4+ neutrophil percentage was not correlated with gestational age or developmental age. Neonates with sepsis had a higher percentage than those without the condition, 66.9% (IQR 24.3–76.9%) versus 21.5% (IQR 10.6–34.7%), respectively, p = 0.0003. At birth, a high percentage of OLFM4+ neutrophils was associated with severe chorioamnionitis at 49.1% (IQR 28.2–61.5%) compared to those without it at 13.7% (IQR 7.7–26.3%), p < 0.0001. Among neonates without sepsis, the percentages of OLFM4+ neutrophils were lower in the BPD/early death group compared to those without BPD, 11.8% (IQR 6.3–29.0%) versus 32.5% (IQR 18.5–46.1%), p = 0.003, and this retained significance in a multiple logistic regression model that included gestational age, birthweight, and race. Conclusion: This is the first study describing OLFM4+ neutrophils in neonates and it shows that this neutrophil subpopulation is not influenced by gestational age but is elevated in inflammatory conditions such as sepsis and severe chorioamnionitis, and lower percentage at birth is associated with developing bronchopulmonary dysplasia.

Preterm Infant Outcomes Following COVID-19 Lockdowns in Melbourne, Australia.

Background Community lockdowns during the coronavirus disease 2019 (COVID-19) pandemic may influence preterm birth rates, but mechanisms are unclear. Methods We compared neonatal outcomes of preterm infants born to mothers exposed to community lockdowns in 2020 (exposed group) to those born in 2019 (control group). Main outcome studied was composite of significant neonatal morbidity or death. Results Median gestational age was 35 + 4 weeks (295 infants, exposed group) vs. 35 + 0 weeks (347 infants, control group) (p = 0.108). The main outcome occurred in 36/295 (12.2%) infants in exposed group vs. 46/347 (13.3%) in control group (p = 0.69). Continuous positive airway pressure (CPAP) use, jaundice requiring phototherapy, hypoglycaemia requiring treatment, early neonatal white cell and neutrophil counts were significantly reduced in the exposed group. Conclusions COVID-19 community lockdowns did not alter composite neonatal outcomes in preterm infants, but reduced rates of some common outcomes as well as early neonatal inflammatory markers.

1012. Neutrophil Function Enhanced by Recombinant Interferon-Gamma in Newborns

BackgroundFunctional differences exist between neonatal and adult neutrophils. The incidence of infection is higher in preterm infants, and the severity of the immune impairment on the neonatal neutrophils is inversely related to gestational age. In order to recognize and combat life-threatening infections, neonates rely predominantly on the innate immune system.Neutrophils are an essential component of innate immunity, and they are the first responders against bacterial and fungal infections. Sepsis continues to be a prominent cause of neonatal mortality, especially among preterm infants. Recombinant interferon-gamma (IFNγ) effects on the immune system have included the upregulation of TLRs expression and stimulation of phagocytosis. They have been shown to reduce severe infections in children with chronic granulomatous disease.MethodsAfter the protocol was IRB approved, we enrolled term infants in their first 48 hours of life (Table 1). We then obtained free flow whole-blood samples through venipuncture from the cephalic vein. Samples were incubated with and without IFNγ for 24 hours. Isolation of unperturbed neutrophils using immunomagnetics was performed for a final concentration of 1x106/mL. We then assessed the neutrophil-bacterial interaction using fluorescent GFP-Staphylococcus aureus, and quantified neutrophil killing function on a novel assay involving fibrin matrix as a more physiologic and three-dimensional (3D) environment than standard in vitro or culture-based assays. We evaluated normalized progressive ratios, 20μL/80μL, 30μL/70μL, 40μL/60μL of Neutrophil/GFP-S aureus respectively.Table 1 ResultsOn the 20 samples, we observed significant differences demonstrating a considerably enhanced phagocytosis on those samples with the addition of IFNγ(p< 0.0001, Table 2 and Figures 1-3). ConclusionThe phagocytic ability of neonatal neutrophils was greatly enhanced by the addition of IFNγ in term infant blood. Ongoing work will determine whether this remains true for preterm-infant neutrophils and will further delineate mechanisms of these differences. We recognized an opportunity for interferon-based immunomodulation in certain situations on this population at high risk for invasive bacterial infections. Disclosures Ricardo Castillo-Galvan, MD MPH, Karius Inc. (Consultant) Isaac Thomsen, MD, MSCI, Horizon Therapeutics (Consultant)

Placental HTRA1 cleaves α1-antitrypsin to generate a NET-inhibitory peptide

Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (polymorphonuclear leukocytes [PMNs]) fail to form NETs due to circulating NET-inhibitory peptides (NIPs), cleavage fragments of α1-antitrypsin (A1AT). How fetal and neonatal blood NIPs are generated remains unknown, however. The placenta expresses high-temperature requirement serine protease A1 (HTRA1) during fetal development, which can cleave A1AT. We hypothesized that placentally expressed HTRA1 regulates the formation of NIPs and that NET competency changed in PMNs isolated from neonatal HTRA1 knockout mice (HTRA1−/−). We found that umbilical cord blood plasma has elevated HTRA1 levels compared with adult plasma and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of molecular weight similar to previously identified NIPs that block NET formation by adult neutrophils. We showed that neonatal mouse pup plasma contains A1AT fragments that inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. Lipopolysaccharide-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation after birth. However, plasma from HTRA1−/− pups had no detectable NIPs, and PMNs from HTRA1−/− pups became NET competent earlier after birth compared with HTRA1+/+ littermate controls. Finally, in the cecal slurry model of neonatal sepsis, A1ATM383S-CF improved survival in C57BL/6 pups by preventing pathogenic NET formation. Our data indicate that placentally expressed HTRA1 is a serine protease that cleaves A1AT in utero to generate NIPs that regulate NET formation by human and mouse PMNs.

Developmental adaptations of neonatal neutrophils (review)

Neutrophils are crucial components of the innate immunity. Differences exist in the physiological, phenotypic, and functional characteristics between neonatal and adult neutrophils. The severity of these changes is inversely proportional to gestational age, which indicates the dynamic development of these cells throughout pregnancy. Therefore, functional insufficiency of neonatal neutrophils is associated with an extremely high risk of developing neonatal infections and sepsis in infants born premature. Neonatal neutrophils are adapted to conditions that prevent unwanted triggering of proinflammatory factors. In addition, suppression of neutrophil functions is necessary to create a healthy microbiome in the postpartum period; however, it can be inhibit the development of a response to pathogenic organisms. Mechanisms underlying the normal transition of functionally limited neutrophils, capable of fully resisting pathogenic microorganisms, have not been established. This review presents features of neutrophil ontogenetic adaptation to intrauterine conditions and early neonatal period and their potential role in neonatal pathology.

The Expression of CD64 and CD11b Neutrophils in Neonatal Necrotizing Enterocolitis

Objectives: To investigate the expression of CD64 and CD11b neutrophils in neonates with necrotizing enterocolitis (NEC) and to evaluate the diagnostic value of CD64 and CD11b in identifying NEC in neonates. Methods: A total of 138 newborns, admitted to our neonatal intensive care unit from October 2018 to March 2020, were recruited in this study. The subjects were divided into the NEC and control groups, according to the method of case-control. The expression of CD64, CD11b, and C-reactive protein (CRP) was measured in the peripheral blood, the sensitivity and specificity of each index in diagnosing NEC were compared, and their diagnostic value was examined. Results: The corrected gestational age of the NEC and control groups was 32.29 ± 1.85 and 33.24 ± 1.98 weeks, respectively. The mean birth weight of the two groups was 1654 ± 373 and 1746 ± 400 g, respectively. Also, the mean age at sampling was 11.6 ± 3.11 and 11.7 ± 3.14 days in the NEC and control groups, respectively. Gender, corrected gestational age, mean birth weight, and sampling age were not significantly different between the groups (P &gt; 0.05). The CD64 and CD11b expression levels were higher in the NEC group, compared to the control group (P &lt; 0.01). Comparison of the NEC group by stage showed that the CD64 and CD11b indices were the highest in stage III, followed by stage II and stage I (P = 0.01). However, both indices showed a downward trend during the recovery period (P &lt; 0.01). A CD64 level ≥ 0.71, a CD11b level of 1.94, and a CRP level ≥ 7.38 were considered as the best diagnostic criteria. The highest exponential sensitivity (82.7%) was attributed to the CD64 index, followed by CD11b (50.8%) and CRP (39.1%). The highest sensitivity (89%) was obtained by a combination of two indices (one positive index); also, when both indices were positive, the highest specificity (87%) was reported. The sensitivity of combined indices for NEC diagnosis in stage I, II and III were 84.4%, 95.7%, and 92.9%, respectively. Conclusions: The expression levels of CD64 and CD11b neutrophils in neonatal NEC increased and was associated with the severity of NEC. Monitoring of changes in the two indices had a clinical value in the diagnosis of NEC. In conclusion, the combined use of these two indices can be more valuable in the diagnosis of neonatal NEC.

Colostrum fails to prevent bovine/camelid neonatal neutrophil damage from AFB1

Exposure to environmental toxicants that affect the immune system and overall health of many mammals is mostly unavoidable. One of the more common substances is the mycotoxins, especially carcinogenic aflatoxin (AF)B1 which also causes immune suppression/dysregulation in exposed hosts. The present study analyzed the effects of naturally occurring levels of AFB1 on apoptosis of healthy bovine and camelid neonatal neutrophils (PMN) that were isolated both before and after host consumption of colostrum. Cells from bovine and camel neonates (n = 12 sets of PMN/mammal/timepoint) were exposed for 24 h to a low level of AFB1 (i.e. 10 ng AFB1/ml) and then intracellular ATP content and caspase-3, -7, and -9 activities (determined by bioluminescence) were assessed. The results indicated a significant lessening of intracellular ATP content and equivalents of luminescence intensity in AFB1-treated PMN in all studied samples, i.e. isolated pre-and post-colostrum consumption. In contrast, caspase-3, -7, and -9 activities in both pre- and post-colostrum consumption bovine and camelid PMN were noticeably increased (∼>2-fold). The damaging effects of AFB1 were more pronounced in bovine neonate PMN than in camelid ones. These results showed that camelid or bovine neonatal PMN collected pre- and post-colostrum are sensitive (moreso after consumption) to naturally occurring levels of AFB1. While merits of colostrum are well known, its failure to mitigate toxic effects of AFB1 in what would translate into a critical period in the development of immune competence (i.e. during the first few days of life in bovine and camelid calves) is surprising. The observed in vitro toxicities can help clarify underlying mechanisms of immune disorders caused by AFs in animals/humans.

Short communication: Effect of granulocyte-macrophage colony-stimulating factor on neonatal calf peripheral blood neutrophil function in vitro

Neutrophils are innate immunity cells that represent the first line of cellular defense against invading pathogens. Dairy calves, however, experience neutrophil dysfunction during the first weeks of age, contributing to increased disease susceptibility during this period. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that improves neutrophil function in neonates of other species and mature cows. However, its capability to improve neonatal calf neutrophil function is unknown. Therefore, our objective was to evaluate the effect of GM-CSF on the functional capabilities of neutrophils of neonatal calves in vitro. We hypothesized that supplementation of neonatal neutrophils with GM-CSF would increase microbicidal functions to levels comparable with those of mature immunocompetent cattle. For this, we isolated blood neutrophils from 12 healthy 2- to 3-d-old Holstein calves, and neutrophils from 6 mid-lactation Holstein cows were used as a reference of robust neutrophil function. Subsequently, neutrophils from both calves and cattle were incubated for 9 h with 4 concentrations (0, 0.005, 0.05, or 0.5 µg/mL) of GM-CSF, and microbicidal function of neutrophils was assessed in terms of phagocytosis, respiratory burst, myeloperoxidase (MPO) activity, and extracellular trap formation. Mixed models with Tukey pairwise comparisons were used to identify differences among treatment and age groups. Supplementation of GM-CSF in vitro increased phagocytosis and MPO activity of calf and cow neutrophils, although not in a concentration-dependent manner. Respiratory burst and extracellular trap formation were not affected by GM-CSF supplementation. All the microbicidal capacity functions assessed were lower in neutrophils from calves, but supplementation with GM-CSF increased phagocytosis and MPO activity of calf neutrophils to levels comparable with unsupplemented cow neutrophils. Collectively, our results demonstrated that in vitro supplementation of calf neutrophils with GM-CSF enhanced some functional microbicidal capabilities to levels comparable with immunocompetent cattle. Hence, it may be possible to augment the functional capacity of calf neutrophils in vivo through the therapeutic application of GM-CSF and consequently enhance calves' resistance to infections. This should be tested in future in vivo studies.

Interleukin-6 elevation in healthy neonates.

This study aimed to identify peripartum and neonatal factors associated with elevated Interleukin-6 levels in the cord blood of neonates without clinical signs of an infection. We conducted a prospective single-center study with healthy term and preterm neonates between March and November 2017. We investigated correlations between 21 peripartum factors and neonatal IL-6 concentrations. Four hundred and seventy-one infants (GA: 32.9-42.3 weeks) were included. The risk for elevated neonatal IL-6 levels was 3.1 to 4.5-fold increased in the presence of either peripartum maternal temperature >37.5 °C (p = 0.012), duration of labor >12 h (p < 0.001), vaginal delivery (p < 0.001), or neonatal neutrophils >8 × 109 cells/L (p < 0.001). The results indicate that a considerable number of neonates with elevated IL-6 levels can sufficiently cope with an exposition to substantial perinatal stress or intrauterine inflammation and do not require postnatal antibiotic treatment.

Gestation-wise Reference Ranges of Neutrophil Counts in Indian Newborns

ObjectivesBlood counts are commonly performed tests in neonatal intensive care units with the results having various clinical ramifications. Interpreting blood counts as normal or abnormal requires reference ranges as per gestation. Studies on reference ranges for neonatal neutrophil counts are already scarce, and data is lacking in the Asian context. We sought to formulate gestation-wise reference ranges of neutrophil counts in an Indian setting.MethodsHealthy, newborn babies of either gender, aged between 30 to 41 weeks gestation were included in the study. Gestational age was corroborated through first trimester dating scan and postnatally by the New Ballard Score. Single venous blood samples were drawn on day three and day five for estimation of total leukocyte count, differential count (neutrophils, lymphocytes), and peripheral blood smear examination.ResultsWe evaluated the data of 420 newborns. The normative values were compiled week-wise for gestational ages of 30 to 41 weeks at birth. We observed a clustering of neutrophil count values below 8000 cells/μL on day three and below 5000 cells/μL on day five. No gender-based differences in counts were observed. We were able to generate reference range curves for neutrophil counts as per gestational age.ConclusionsThe absolute neutrophil counts of term and preterm Indian newborns are higher than the values depicted in the standard reference chart used currently. This indicates that a different standard chart as per gestation should be used in Indo-Asian countries to differentiate ‘normal’ from ‘abnormal’.

Local anaesthetics upregulate nitric oxide generation in cord blood and adult human neutrophils

Nitric oxide (NO) generation by systemic neonatal neutrophils is not clarified. It is also not known whether local anaesthetics (LAs) transferred to the fetal systemic circulation following maternal epidural blockade may affect this process. In the present study, NO generation was evaluated in neutrophils from cord blood (CB, n = 11) and adult blood (n = 10) following exposure to bupivacaine (0.0005, 0.005, 1 mM), lidocaine (0.002, 0.02, 4 mM) and ropivacaine (0.0007, 0.007, 1.4 mM) using flow cytometry, as well as indirectly by determining nitrite concentrations in cell incubation media. To determine the role of NO synthase (NOS) isoforms in NO generation following exposure to LAs, experiments were repeated in the presence of the NOS inhibitors, NG-nitro-L-arginine methyl ester and aminoguanidine; in addition, the expression of NOS isoforms was analysed. CB neutrophils produced less NO than adult neutrophils. LAs, especially ropivacaine and lidocaine, stimulated neutrophil NO generation, but in CB neutrophils this effect was negligible at clinically relevant drug concentrations. A mechanism involving NOS activity was responsible for the observed phenomena. In conclusion, LAs are able to upregulate neutrophil NO production, but in neonates this effect is likely to be clinically insignificant.

Estimation of Neonatal Intestinal Perforation Associated with Necrotizing Enterocolitis by Machine Learning Reveals New Key Factors.

Intestinal perforation (IP) associated with necrotizing enterocolitis (NEC) is one of the leading causes of mortality in premature neonates; with major nutritional and neurodevelopmental sequelae. Since predicting which neonates will develop perforation is still challenging; clinicians might benefit considerably with an early diagnosis tool and the identification of critical factors. The aim of this study was to forecast IP related to NEC and to investigate the predictive quality of variables; based on a machine learning-based technique. The Back-propagation neural network was used to train and test the models with a dataset constructed from medical records of the NICU; with birth and hospitalization maternal and neonatal clinical; feeding and laboratory parameters; as input variables. The outcome of the models was diagnosis: (1) IP associated with NEC; (2) NEC or (3) control (neither IP nor NEC). Models accurately estimated IP with good performances; the regression coefficients between the experimental and predicted data were R2 > 0.97. Critical variables for IP prediction were identified: neonatal platelets and neutrophils; orotracheal intubation; birth weight; sex; arterial blood gas parameters (pCO2 and HCO3); gestational age; use of fortifier; patent ductus arteriosus; maternal age and maternal morbidity. These models may allow quality improvement in medical practice.

Impact of toll-like receptor 4 stimulation on human neonatal neutrophil spontaneous migration, transcriptomics, and cytokine production.

Preterm neonates have reduced spontaneous neutrophil migration compared to term neonates and adults in the absence and presence of TLR4 stimulation. Preterm and term neonates have reduced neutrophil velocities compared to adults in the absence of TLR4 stimulation but no difference in the presence of TLR4 stimulation. Unique transcriptomic response to TLR4 stimulation is observed in neutrophils from preterm neonates, term neonates, and adults. TLR4 stimulation produces an age-specific cytokine response.