B-Cell-Activating Factor Contributes to Elevation of the Content of Regulatory B Cells in Neonatal Sepsis.

We studied the role of B cell-activating factor (BAFF) in PI3K/AKT/mTOR signaling pathway in promoting proliferation and maintaining survival of regulatory B lymphocytes (Breg) in newborns with sepsis. The peripheral blood samples were collected from preterm neonates (n=40) diagnosed with sepsis on the day of diagnosis and on days 7, 14, and 21 after diagnosis, as well as from the matched preterm neonates without sepsis (n=40; control group). The peripheral blood mononuclear cells and B cells were isolated, cultured, and stimulated with LPS and immunostimulant CpG-oligodeoxynucleotide (CpG-ODN). Proliferation and differentiation of B-cells into CD19+CD24hiCD38hi Breg cells and the role of the PI3K/AKT/mTOR signaling pathway in these processes were studied by flow cytometry, real-time quantitative reverse transcription PCR (qRT-PCR), and Western blotting. BAFF levels in the peripheral blood of neonates with sepsis were significantly increased at one week after diagnosis in parallel with increasing trend of expression of BAFF receptor. When applied with LPS and CpG-ODN, BAFF promoted differentiation of B cells into CD19+CD24hiCD38hi Breg cells. Phosphorylation of 4E-BP1 factor and 70S6K kinase located downstream in PI3K/AKT/mTOR signaling pathway was significantly up-regulated when stimulated with BAFF in combination with LPS and CpG-ODN. Thus, increased level of BAFF activates PI3K/AKT/mTOR signaling pathway and induces in vitro differentiation of peripheral blood B cells into CD19+CD24hiCD38hi Breg cells.

A20 and the noncanonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny.

Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.

Nucleotide receptors mediate protection against neonatal sepsis and meningitis caused by alpha-hemolysin expressing Escherichia coli K1.

Neonatal meningitis-associated Escherichia coli (NMEC) is among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here, we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha-hemolysin (HlyA)-expressing NMEC (HlyA+ ) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA- NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA- NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activates P2XRs which in turn confers macrophage-dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.

Survival and Pulmonary Injury After Neonatal Sepsis: PD1/PDL1's Contributions to Mouse and Human Immunopathology.

Morbidity and mortality associated with neonatal sepsis remains a healthcare crisis. PD1-/- neonatal mice endured experimental sepsis, in the form of cecal slurry (CS), and showed improved rates of survival compared to wildtype (WT) counterparts. End-organ injury, particularly of the lung, contributes to the devastation set forth by neonatal sepsis. PDL1-/- neonatal mice, in contrast to PD1-/- neonatal mice did not have a significant improvement in survival after CS. Because of this, we focused subsequent studies on the impact of PD1 gene deficiency on lung injury. Here, we observed that at 24 h post-CS (but not at 4 or 12 h) there was a marked increase in pulmonary edema (PE), neutrophil influx, myeloperoxidase (MPO) levels, and cytokine expression sham (Sh) WT mice. Regarding pulmonary endothelial cell (EC) adhesion molecule expression, we observed that Zona occludens-1 (ZO-1) within the cell shifted from a membranous location to a peri-nuclear location after CS in WT murine cultured ECs at 24hrs, but remained membranous among PD1-/- lungs. To expand the scope of this inquiry, we investigated human neonatal lung tissue. We observed that the lungs of human newborns exposed to intrauterine infection had significantly higher numbers of PD1+ cells compared to specimens who died from non-infectious causes. Together, these data suggest that PD1/PDL1, a pathway typically thought to govern adaptive immune processes in adult animals, can modulate the largely innate neonatal pulmonary immune response to experimental septic insult. The potential future significance of this area of study includes that PD1/PDL1 checkpoint proteins may be viable therapeutic targets in the septic neonate.

Utility of volatile organic compounds as a diagnostic tool in preterm infants.

BACKGROUND: Volatile organic compounds (VOCs) are hydrocarbons that originate within different healthy and diseased tissues. VOCs can be secreted into the circulation and then excreted in the urine and faeces. In the lungs, VOCs are locally produced and can be detected in exhaled breath. VOCs can be identified using non-invasive techniques, which make their use in preterm infants safe and desirable. METHODS: A systematic search of the literature in PubMed, Embase and Web of Science was conducted looking for VOCs techniques and diagnostic performance in preterm infants. A total of 50 articles identified with only seven papers were included in the final analysis in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). RESULTS: VOCs could diagnose necrotising enterocolitis up to 4 days before a clinical diagnosis; for late onset sepsis, up to 3 days before; and for bronchopulmonary dysplasia, up to 2 weeks before. In addition to these diagnostic uses, VOCs analysis could also distinguish breastfed from formula-fed preterm neonates in the first month of life. CONCLUSION: VOCs analysis is a non-invasive tool that makes the use in preterm infants of preference. VOCs analytic techniques require more research and consensus between researchers to overcome their limitations. IMPACT: Volatile organic compounds are hydrocarbons that can separate between healthy and diseased states in preterm infants. Biomarker panels developed from volatile organic compounds are potential diagnostic tools. The non-invasive nature of acquiring volatile organic compounds markers make it desirable in the paediatric patients. Research into exact chemical components of the volatile organic compounds can inform about the pathophysiology of disease in preterm infants. More robust longitudinal studies with repeated experiments are required before volatile organic compounds can be applied in clinical practice.

Maternal activation of the EGFR prevents translocation of gut-residing pathogenic Escherichia coli in a model of late-onset neonatal sepsis.

Late-onset sepsis (LOS) is a highly consequential complication of preterm birth and is defined by a positive blood culture obtained after 72 h of age. The causative bacteria can be found in patients' intestinal tracts days before dissemination, and cohort studies suggest reduced LOS risk in breastfed preterm infants through unknown mechanisms. Reduced concentrations of epidermal growth factor (EGF) of maternal origin within the intestinal tract of mice correlated to the translocation of a gut-resident human pathogen Escherichia coli, which spreads systemically and caused a rapid, fatal disease in pups. Translocation of Escherichia coli was associated with the formation of colonic goblet cell-associated antigen passages (GAPs), which translocate enteric bacteria across the intestinal epithelium. Thus, maternally derived EGF, and potentially other EGFR ligands, prevents dissemination of a gut-resident pathogen by inhibiting goblet cell-mediated bacterial translocation. Through manipulation of maternally derived EGF and alteration of the earliest gut defenses, we have developed an animal model of pathogen dissemination which recapitulates gut-origin neonatal LOS.

Neutrophil Volume, conductivity and scatter (VCS) as a screening tool in neonatal sepsis.

The initial evaluation of a suspected sepsis in a neonate is always challenging. There are many methods to screen a neonate with suspected sepsis. One of newer method is to assess the changes in neutrophil volume conductivity and scatter. The objective of this study was to establish changes in Neutrophil volume conductivity scatter (VCS) in neonatal sepsis and to determine appropriate cut off levels using receiver operating characteristic (ROC) curves. Neonates with suspected sepsis were evaluated with blood counts, culture and neutrophil VCS parameters. Based on these parameters neonates were classified into sepsis group (Blood culture positive), Probable sepsis group (clinical course consistent with sepsis and positive sepsis screen and negative blood culture), No sepsis group (Clinical course not suggestive of sepsis with negative sepsis screen and blood culture). A total of 304 neonates were included in the study of which 144 were in sepsis group and 160 in no sepsis group respectively. Among the neutrophil VCS parameters there was significant difference between the groups with respect to mean neutrophil volume (MNV) and volume distribution width (VDW) (180 vs 163 vs 150) (p < 0.01). MNV and VDW had good sensitivity (95%, 82%) and specificity (86%, 74%) for diagnosis of sepsis. In conclusion, Neutrophil VCS parameters, especially MNV, can be incorporated with other sepsis screen parameters in diagnosis of neonatal sepsis.

Progranulin as a novel biomarker in diagnosis of early-onset neonatal sepsis.

BACKGROUND: Infections are leading causes of morbidity and mortality in neonates and may also have severe long-term consequences. As early diagnosis of neonatal sepsis improves prognosis, identification of new or complementary biomarkers is of great importance. In this study, we have evaluated the diagnostic value of progranulin (PGRN) in early-onset neonatal sepsis (EOS) and compare its effectiveness with other commonly used biomarkers, such as procalcitonin (PCT) and C-reactive protein (CRP). METHODS: A total of 121 infants with gestational age of >34 weeks admitted with suspected EOS were included in this study. Before initiating therapy, blood samples for whole blood count, CRP, PCT and PGRN were obtained from all neonates. Receiver-operating characteristic (ROC) curve and multivariate logistic regression analyses were performed. RESULTS: Serum PGRN level of infected group was significantly higher than uninfected group (median 47.72 vs. 37.86 ng/ml, respectively; Mann-Whitney p < 0.0001). The ROC area under the curve (AUC) was 0.786 [95% confidence interval (CI) 0.706-0.867; p < 0.0001] for PGRN, 0.699 (95% CI 0.601-0.797; p = 0.0001) for age adjusted PCT, and 0.673 (95% CI 0.573-0.773; p = 0.0007) for CRP. With a cut-off value of 37.89 ng/ml, the diagnostic sensitivity and negative predictive value of PGRN were 94.34% and 91.7%, respectively. PGRN could significantly predict EOS independently of PCT (p < 0.0001), and the combined use of PGRN and PCT could significantly improve diagnostic performance for EOS (0.806; 95% CI 0.73-0.88; p < 0.0001), with a specificity of 89.06% and a positive predictive value of 81.10%. CONCLUSIONS: PGRN may be used as a promising biomarker for the diagnosis of EOS, and the combined use of PGRN and PCT could improve the diagnosis of sepsis.

C23, an oligopeptide derived from cold-inducible RNA-binding protein, suppresses inflammation and reduces lung injury in neonatal sepsis.

INTRODUCTION: Neonatal sepsis remains a leading cause of infant mortality. Cold-inducible RNA binding protein (CIRP) is an inflammatory mediator that induces TNF-α production in macrophages. C23 is a CIRP-derived peptide that blocks CIRP from binding its receptor. We therefore hypothesized that treatment with C23 reduces systemic inflammation and protects the lungs in neonatal sepsis. METHODS: Sepsis was induced in C56BL/6 mouse pups (5-7 days) by intraperitoneal injection of adult cecal slurry (0.525 mg/g body weight, LD100). One hour later pups received retroorbital injection of C23 (8 mg/kg) or vehicle (normal saline). Ten hours after sepsis induction, blood and tissues were collected for analysis. RESULTS: C23 treatment resulted in a 58% and 69% reduction in serum levels of proinflammatory cytokines IL-6 and IL-1ß, respectively, and a 40% and 45% reduction of AST and LDH, as compared to vehicle-treated septic pups. In the lungs, C23 treatment reduced expression of cytokines IL-6 and IL-1ß by 78% and 74%. In addition, the mRNA level of neutrophil chemoattractants KC and MIP-2 was reduced by 84% and 74%, respectively. These results corresponded to a reduction in histologic lung injury score. Vehicle-treated pups scored 0.49 ±â€¯0.19, while C23 treatment reduced scores to 0.29 ±â€¯0.12 (p < 0.05; Max = 1). Apoptosis in the lungs, measured by TUNEL assay, was also decreased by 53% with C23 treatment (p < 0.05). CONCLUSIONS: Inhibition of CIRP with C23 treatment is protective in septic neonatal mice as demonstrated by reduced inflammatory markers systemically and in the lung. Therefore, C23 has promising therapeutic potential in treatment of neonatal sepsis. LEVEL OF EVIDENCE: Level I.

Assessment of oxidative damage and enzymatic antioxidant system activity on the umbilical cord blood and saliva from preterm newborns with risk factors for early-onset neonatal sepsis

SUMMARY BACKGROUND: To determine the concentration of the Lipid Peroxidation Marker: Malondialdehyde (MDA), and Antioxidant Markers: Superoxide Dismutase (SOD), Glutathione Peroxidase (GPX), Catalase (CAL) in umbilical cord blood and in unstimulated saliva in the first 24 and 48 hours of life in the PTNB of mothers with and without risk factors for early-onset neonatal sepsis. METHODS: Cross-sectional study with the signing of informed consent by the pregnant women and application of a standard questionnaire classifying the PTNB in Group 1 or 2. RESULTS: Twenty-one PTNB were studied. Regarding gender, birth weight, need for oxygen, use of phototherapy, diagnosis of assumed sepsis, presence of fetal distress, number of pregnancies, type of delivery, use of corticosteroids, premature rupture of membranes, maternal fever, chorioamnionitis, APGAR at the 5th and 10th minute of life. Statistical analysis was performed with the Mann-Whitney test (p = 0.019) on the GPX variable of umbilical cord blood in the group of mothers with risk factors for early-onset neonatal sepsis. There was no statistical difference in the MDA, SOD, and CAT variables of the group with risk factors and in any variable of the group without risk factors. CONCLUSION: There was an increase of the GPX concentration in the blood from the umbilical vein in the group with risk factors for early-onset neonatal sepsis. There was no statistical significance in the comparison of saliva and umbilical cord blood. There was no statistically significant difference in MDA, SOD, CAT.

RESUMO OBJETIVOS: Determinar a concentração do marcador de peroxidação lipídica: Malondialdeído (MDA) e dos marcadores antioxidantes: Superóxido Dismutase (SOD), Glutationa Peroxidase (GPX), Catalase (CAL) no sangue do cordão umbilical e na saliva não estimulada nas primeiras 24 e 48 horas de vida nos RNPT de mães com e sem fatores de risco para sepse neonatal precoce. METODOLOGIA: Estudo transversal com a assinatura do termo de consentimento livre esclarecido pela gestante e aplicação de um questionário padrão classificando o RNPT no Grupo 1 ou 2. RESULTADOS: Foram estudados 21 RNPT. Quanto ao gênero, peso ao nascimento, necessidade de oxigênio, uso de fototerapia, diagnóstico de sepse presumida, presença de sofrimento fetal, número de gestações, tipo de parto, uso de corticoide, rotura prematura de membranas, a presença de febre materna, a presença de corioamnionite, Apgar no 50 e 100 minuto de vida, a análise estatística foi feita com o teste de Mann-Whitney (p=0,019) na váriável GPX do sangue do cordão umbilical no grupo das mães com fatores de risco para sepse neonatal precoce. Não houve diferença estatística nas outras variáveis MDA, SOD, CAT do grupo com fatores de risco e em nenhuma variável do grupo sem fatores de risco. CONCLUSÃO: O aumento de duas vezes a concentração da GPX no sangue da veia umbilical dos RNPT do grupo das mães com fatores de risco para sepse neonatal precoce. Sem significância estatística na comparação entre a saliva e o sangue do cordão umbilical. Não houve diferença estatisticamente significante nas variáveis MDA, SOD e CAT.

Deficiency of receptor-interacting protein kinase 3 (RIPK3) attenuates inflammation and organ injury in neonatal sepsis.

INTRODUCTION: Sepsis is the third leading cause of morbidity and mortality in neonates. Sepsis in neonates is characterized as the systemic inflammation owing to infection within the first 28days after birth. The molecular mechanism causing the exaggerated inflammation phenotype in neonates has not been completely elucidated. Receptor interacting protein kinase 3 (RIPK3) is a protein identified as a mediator in programmed necrosis or necroptosis. We hypothesize that RIPK3 could be responsible for the inflammatory response in neonates and that deficiency in the RIPK3 protein attenuates inflammation and organ injury in neonatal sepsis. METHODS: Male and female C57BL6 wild-type (WT) and RIPK3 knock-out (KO) newborn mice aged 5-7days (3-4g body weight) were injected intraperitoneally with 0.9mg/g cecal slurry (CS). At 10h after injection, the newborns were euthanized and blood, the lungs and gut tissues were collected. RESULTS: At 10h after CS injection, serum cytokines IL-6 and IL-1ß in the WT mice were increased by 511- and 43-fold whereas in KO mice, these levels were increased by 166-fold and 22-fold, respectively. Lung IL-1ß in the WT mice increased by 7-fold after CS injection whereas only a 4-fold increase was seen in the KO mice. In the lungs of CS injected KO mice, the injury score, MIP-2 mRNA, myeloperoxidase (MPO) activity and TUNEL staining were significantly reduced by 76%, 70%, 26% and 74%, respectively compared to the CS WT mice. Gut TUNEL staining was also reduced by 80%. CONCLUSION: The deficiency in RIPK3 attenuated serum and lung cytokines, lung injury and neutrophil infiltration and lung and gut apoptosis. These data suggest that RIPK3, in part, is responsible for the systemic inflammatory response in neonatal sepsis.

Deficiency in milk fat globule-epidermal growth factor-factor 8 exacerbates organ injury and mortality in neonatal sepsis.

INTRODUCTION: Neonatal sepsis is a systemic inflammation occurring in neonates because of a proven infection within the first 28days of birth. It is the third leading cause of morbidity and mortality in the newborns. The mechanism(s) underlying the systemic inflammation in neonatal sepsis has not been completely understood. We hypothesize that the deficiency of milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a protein commonly found in human milk, could be responsible for the increased inflammatory response leading to morbidity and mortality in neonatal sepsis. METHODS: Male and female newborn mice aged 5-7days were injected intraperitoneally with 0.9mg/g body weight cecal slurry (CS). At 10h after CS injection, they were euthanized, and blood, lungs and gut tissues were obtained for further analyses. Control newborn mice underwent similar procedures with the exception of the CS injection. In duplicate newborn mice after CS injection, they were returned to their respective cages with their mothers and were closely monitored for 7days and survival rate recorded. RESULTS: At 10h after CS injection, serum LDH in the MFG-E8 knockout (KO) newborn mice was significantly increased by 58% and serum IL-6, IL-1ß and TNF-α in the MFG-E8KO newborn mice were also significantly increased by 56%, 65%, and 105%, respectively, from wild type (WT) newborn mice. There were no significant difference between WT control and MFG-E8 control newborn mice. The lung architecture was severely damaged and a significant 162% increase in injury score was observed in the CS MFG-E8KO newborn mice. The MPO, TUNEL staining, and cytokine levels in the lungs and the intestine in CS MFG-E8KO newborn mice were significantly increased from CS WT newborn mice. Similarly, intestinal integrity was also compromised in the CS MFG-E8KO newborn mice. In a survival study, while the mortality rate within 7days was only 29% in the CS WT newborn mice, 80% of the CS MFG-E8KO newborn mice died during the same time period with the majority of mortality occurring within 48h. CONCLUSION: The deficiency in MFG-E8 caused increases in inflammation, tissue injury, neutrophil infiltration and apoptosis, which led to morbidity and mortality in murine neonatal sepsis. These studies suggest that MFG-E8 has a protective role in fighting against neonatal sepsis.