Lactobacillus Acidophilus/Bifidobacterium Infantis Probiotics Are Beneficial to Extremely Low Gestational Age Infants Fed Human Milk.

Objective: To evaluate the nutrition-related effects of prophylactic Lactobacillus acidophilus/Bifidobacterium infantis probiotics on the outcomes of preterm infants <29 weeks of gestation that receive human milk and/or formula nutrition. We hypothesize that human-milk-fed infants benefit from probiotics in terms of sepsis prevention and growth. METHODS: We performed an observational study of the German Neonatal Network (GNN) over a period of six years, between 1 January, 2013 and 31 December, 2018. Prophylactic probiotic use of L. acidophilus/B. infantis was evaluated in preterm infants <29 weeks of gestation (n = 7516) in subgroups stratified to feeding type: (I) Exclusively human milk (HM) of own mother and/or donors (HM group, n = 1568), (II) HM of own mother and/or donor and formula (Mix group, n = 5221), and (III) exclusive exposure to formula (F group, n = 727). The effect of probiotics on general outcomes and growth was tested in univariate models and adjusted in linear/logistic regression models. RESULTS: 5954 (76.5%) infants received L. acidophilus/B. infantis prophylactically for the prevention of necrotizing enterocolitis (NEC). Probiotic use was associated with improved growth measures in the HM group (e.g., weight gain velocity in g/day: effect size B = 0.224; 95% CI: 2.82-4.35; p < 0.001) but not in the F group (effect size B = -0.06; 95% CI: -3.05-0.28; p = 0.103). The HM group had the lowest incidence of clinical sepsis (34.0%) as compared to the Mix group (35.5%) and the F group (40.0%). Only in the Mix group, probiotic supplementation proved to be protective against clinical sepsis (OR 0.69; 95% CI: 0.59-0.79; p < 0.001). CONCLUSION: Our observational data indicate that the exposure to L. acidophilus/B. infantis probiotics may promote growth in exclusively HM-fed infants as compared to formula-fed infants. To exert a sepsis-preventive effect, probiotics seem to require human milk.

Modulation of S. epidermidis-induced innate immune responses in neonatal whole blood.

BACKGROUND: Coagulase-negative staphylococci (CoNS) such as Staphylococcus epidermidis are highly prevalent pathogens for sepsis in neonates. The interaction between host, environment and pathogenic factors of S. epidermidis are still poorly understood. Our objective was to address the role of several pathogenic factors of S. epidermidis on neonatal cytokine responses and to characterize the influence of three immunomodulatory drugs. METHODS: We performed an ex-vivo model of S. epidermidis sepsis by assessment of blood cytokine production in neonatal whole blood stimulation assays (ELISA). S. epidermidis strains with different characteristics were added as full pathogen to umbilical cord blood cultures and the influence of indomethacin, ibuprofen and furosemide on neonatal immune response to S. epidermidis was evaluated (Flow cytometry). RESULTS: Stimulation with S. epidermidis sepsis strains induced higher IL-6 and IL-10 expression than stimulation with colonization strains. Biofilm formation in clinical isolates was associated with increased IL-10 but not IL-6 levels. In contrast, stimulation with mutant strains for biofilm formation and extracellular virulence factors had no major effect on cytokine expression. Notably, addition of ibuprofen or indomethacin to S. epidermidis inoculated whole blood resulted in mildly increased expression of TNF-α but not IL-6, while frusemide decreased the production of pro-inflammatory cytokines, i.e. IL-6 and IL-8. CONCLUSIONS: The virulence of sepsis strains is coherent with increased cytokine production in our whole-blood in-vitro sepsis model. Biofilm formation and expression of extracellular virulence factors had no major influence on readouts in our setting. It is important to acknowledge that several drugs used in neonatal care have immunomodulatory potential.

Clinical Relevance of Pathogens Detected by Multiplex PCR in Blood of Very-Low-Birth Weight Infants with Suspected Sepsis - Multicentre Study of the German Neonatal Network.

INTRODUCTION: In the German Neonatal Network (GNN) 10% of very-low-birth weight infants (VLBWI) suffer from blood-culture confirmed sepsis, while 30% of VLBWI develop clinical sepsis. Diagnosis of sepsis is a difficult task leading to potential over-treatment with antibiotics. This study aims to investigate whether the results of blood multiplex-PCR (SeptiFast®) for common sepsis pathogens are relevant for clinical decision making when sepsis is suspected in VLBWI. METHODS: We performed a prospective, multi-centre study within the GNN including 133 VLBWI with 214 episodes of suspected late onset sepsis (LOS). In patients with suspected sepsis a multiplex-PCR (LightCycler SeptiFast MGRADE-test®) was performed from 100 µl EDTA blood in addition to center-specific laboratory biomarkers. The attending neonatologist documented whether the PCR-result, which was available after 24 to 48 hrs, had an impact on the choice of antibiotic drugs and duration of therapy. RESULTS: PCR was positive in 110/214 episodes (51%) and blood culture (BC) was positive in 55 episodes (26%). Both methods yielded predominantly coagulase-negative staphylococci (CoNS) followed by Escherichia coli and Staphylococcus aureus. In 214 BC-PCR paired samples concordant results were documented in 126 episodes (59%; n = 32 were concordant pathogen positive results, n = 94 were negative in both methods). In 65 episodes (30%) we found positive PCR results but negative BCs, with CoNS being identified in 43 (66%) of these samples. Multiplex-PCR results influenced clinical decision making in 30% of episodes, specifically in 18% for the choice of antimicrobial therapy and in 22% for the duration of antimicrobial therapy. CONCLUSIONS: Multiplex-PCR results had a moderate impact on clinical management in about one third of LOS-episodes. The main advantage of multiplex-PCR was the rapid detection of pathogens from micro-volume blood samples. In VLBWI limitations include risk of contamination, lack of resistance testing and high costs. The high rate of positive PCR results in episodes of negative BC might lead to overtreatment of infants which is associated with risk of mortality, antibiotic resistance, fungal sepsis and NEC.

Cardiac anaplastic large cell lymphoma in an 8-year old boy.

We report on an 8 year old boy with primary cardiac anaplastic large cell lymphoma (ALCL), in whom the diagnosis was challenging and who was treated with modified chemotherapy without radiation therapy according to the ALCL 99 study protocol [1]. Two years and 4 months after completion of therapy the boy is in complete remission with normal cardiac function.

Differential expression of antimicrobial polypeptides in cord blood samples of preterm and term infants.

AIM: To determine levels of antimicrobial polypeptides (AMP) in cord blood of term and preterm neonates and to investigate influencing factors. METHODS: In a single-centre study, n = 139 preterm infants and n = 36 term infants were included. AMP levels were analysed in supernatants of whole cord blood cultures with a standardised concentration of 5 × 10(6) white blood cells/mL via enzyme-linked immunosorbent assay (ELISA). RESULTS: Lactoferrin, human neutrophil peptides (HNP) 1-3 and bacterial permeability-increasing protein (BPI) expression in cord blood of preterm infants were influenced by the cause of preterm delivery, that is increased levels in infants with clinical amniotic infection. AMP levels also weakly correlated with white blood cell and neutrophil count at birth. In the whole cohort, no association between gestational age or birthweight with AMP levels was found. In the subgroup of infants without clinical amniotic infection (n = 77 preterm infants, n = 36 healthy term infants), we noted a weak correlation between gestational age and lactoferrin, calprotectin and HNP1-3 levels. In addition to that, we observed higher levels of lactoferrin and HNP1-3 in large-for-gestational-age infants. CONCLUSION: Our study confirms that several factors influence cord blood AMP levels which underlines the difficulties of using AMP levels as biomarkers of immunological response.

Risk for late-onset blood-culture proven sepsis in very-low-birth weight infants born small for gestational age: a large multicenter study from the German Neonatal Network.

BACKGROUND: It was the aim of this study to assess whether very-low-birth-weight (VLBW) infants born small for gestational age (SGA; birth weight less than 10th percentile) are at increased risk for late-onset sepsis. METHODS: This was a prospective, multicenter study of the German Neonatal Network including VLBW infants from 23 to < 32 weeks post menstrual age born 2009-2011. Outcomes were compared between VLBW infants born SGA (birth weight less than tenth percentile according to gestational age and gender) and non-SGA infants. The main outcome measure was at least 1 episode of late-onset sepsis defined as blood-culture-confirmed clinical sepsis occurring at ≥ 72 hours of age. RESULTS: 5886 VLBW infants were included. In SGA infants (n = 692), an increased incidence of late-onset sepsis was noted compared with non-SGA infants (20.1% vs. 14.3 %, P < 0.001). This difference was only observed among infants with a gestational age of 27 to < 32 weeks and attributed to sepsis episodes with coagulase-negative staphylococci (12.8% vs. 8.3%, P < 0.001). Different treatment modalities (eg more frequent use of central venous lines) and longer duration of invasive therapies (parenteral nutrition, mechanical ventilation, hospitalization) may account for the increased sepsis risk with coagulase-negative staphylococci in our SGA cohort. In a multivariate logistic regression analysis, higher gestational age [per week; odds ratio (OR): 0.75, 95% confidence interval (CI): 0.72-0.78, P< 0.0001], treatment with antenatal steroids (OR: 0.7, 95% CI: 0.53-0.92, P = 0.01), German descendance (OR: 0.76, 95% CI: 0.63-0.91, P = 0.003) and prophylaxis with glycopeptide antibiotics (OR: 0.64, 95% CI: 0.47-0.87, P = 0.005) were shown to be protective against late-onset sepsis. In contrast, longer duration of parenteral nutrition (per day; OR: 1.016, 95% CI: 1.011-1.021, P < 0.0001) and SGA were found to be risk factors (OR: 1.31, 95% CI: 1.02-1.68, P= 0.03). CONCLUSIONS: SGA contributes to the risk of late-onset sepsis in VLBW infants. Future studies are needed to investigate the underlying pathophysiology to guide individualized preventive measures in this vulnerable subgroup.

Intrauterine growth restriction and the innate immune system in preterm infants of ≤32 weeks gestation.

OBJECTIVE: Intrauterine growth restriction (IUGR) is a well-known cause of adverse neonatal outcomes. As it may have an impact on innate immune responses, we aimed to investigate several parameters of the innate immune response in preterm infants of ≤32 weeks gestation who were small for gestational age (SGA). METHODS: We compared clinical data of SGA (n = 20) and appropriate for gestational age (AGA; n = 124) newborns with a gestational age of ≤32 weeks. We investigated full blood counts at birth and on days 3 and 7 of life and cytokine immune responses in a human whole cord blood assay. RESULTS: SGA preterm infants had a higher risk of the combined outcome mortality or bronchopulmonary dysplasia. Numbers of white blood cells and neutrophils were diminished in SGA infants at birth and on day 3. At birth, platelet counts were also diminished while the number of nucleated red blood cells tended to be elevated in SGA infants. After stimulation of whole blood cell cultures with lipopolysaccharides, the concentrations of interleukin-6 and interleukin-10 were significantly lower in SGA preterm infants compared to AGA infants. CONCLUSIONS: SGA infants differ remarkably from AGA infants in full blood counts and in their ability to mount an immune response. Whether the quantitative deficiency in innate immunity plays a role for adverse outcomes needs to be investigated in larger future trials.

Insulin-like growth factor-I regulates the neonatal immune response in infection and maturation by suppression of IFN-γ.

Disturbances in Insulin-like growth factor-I (IGF-I) and dysregulation in neonatal immune responses have been associated with typical neonatal diseases. Immunosuppression by IGF-I might be a key regulator of neonatal immune responses in infection and maturation. However, information on IGF-I serum levels, IGF-I-receptor (IGF-IR) and effects on functional properties of neonatal immune cells is scarce. Neonatal cord blood samples were stimulated with anti-CD3/anti-CD28, PMA/ionomycin or LPS in a whole-blood assay, while IGF-I serum levels and IGF-I receptor expression were assessed. Furthermore the effect of IGF-I on cytokine expression, apoptosis and the DNA binding activity of AP-1 and NFκB was evaluated. IGF-I serum levels were within normal range. The IGF-I-receptor was present on the surface of cord blood CD3+CD4+lymphocytes and cord blood CD14+monocytes. Upon stimulation the IGF-I-R expression on lymphocytes was significantly upregulated. The addition of IGF-I (100ng/ml) to whole blood cultures inhibited the secretion of IFN-γ from PMA/ionomycin-stimulated cord blood mononuclear cells (CBMC). While IGF-I did not influence apoptosis in our experimental setting, it led to a distinct decrease in anti-CD3/CD28-stimulated DNA binding activity of AP-1 and NFκB. Thus IGF-I may be a key regulator of neonatal immune responses in maturation processes and inflammation by suppressing proinflammatory Th1 responses. Future in vivo studies need to elucidate whether disturbances of the IGF-I serum level and IGF-IR expression are associated with susceptibility for infections and subsequent diseases in neonates.

The effect of hyperglycemia on neonatal immune responses in-vitro.

INTRODUCTION: Acute hyperglycemia is considered as a pro-inflammatory state and is related to an adverse outcome in critically ill adults. Neonates are susceptible to infections and systemic inflammatory response syndrome induced by pro-inflammatory cytokines. This study focuses on the interaction between neonatal glucose homeostasis and the pro-inflammatory cytokine production in term and preterm infants in-vitro. METHODS: We analyzed the pro-inflammatory cytokine production in whole cord blood of term infants (n = 10), preterm infants > 32 weeks (n=16) and preterm infants ≤32 weeks of gestational age (n = 13) and in adult controls (n=14) using an in-vitro sepsis-model. Whole blood was pre-incubated with different concentrations of glucose (0-1000 mg/dl) and insulin (0-62.5 IE/l) and stimulated with lipopolysaccharide. The intracytoplasmatic TNF-α, IL-6, and IL-8 response was measured by flow cytometry. RESULTS: In-vitro hyperglycemia induced a dose-dependent increase of IL-8 in all age groups while TNF-α was demonstrated to be stimulated by glucose in cord blood samples of preterm infants ≤32 weeks of gestational age and term infants. In contrast, insulin showed no significant effects on pro-inflammatory cytokine production in-vitro. CONCLUSION: Acute hyperglycemia may induce pro-inflammatory cytokine responses in neonatal whole blood in-vitro. These data provide a basis for further in-vitro signal transduction studies and in-vivo investigations about the significance of neonatal glucose homeostasis and its impact on long-term outcome of this susceptible patient cohort.

The modulatory effect of lipids and glucose on the neonatal immune response induced by Staphylococcus epidermidis.

BACKGROUND: Parenteral nutrition is an important risk factor for late onset sepsis in neonates. This may be caused by the long-term need of central venous access but also through a potentially modulating effect of lipids and glucose on the immune function. OBJECTIVE: It was the aim of this study to characterize the effect of lipids and glucose on the neonatal immune response in an in vitro Staphylococcus epidermidis sepsis model using whole cord blood of healthy term infants and preterm infants. RESULTS: At the single cell level, IL-6, IL-8 and TNF-α expression of CD14+ cells was significantly increased upon addition of 1% lipids, while the addition of clinically meaningful lipid concentrations had no remarkable effect. When glucose was added to whole cord blood cultures, a dose-dependent effect was demonstrated for IL-8 expression but not for other cytokines. CONCLUSIONS: These in vitro data suggest that the proinflammatory cytokine response to S. epidermidis may be modulated by lipids and glucose. Further studies are needed to investigate whether these findings are applicable to clinical settings and to evaluate the role of cytokine monitoring in infants receiving long-term parenteral nutrition.