Effects of multistrain Bifidobacteria and Lactobacillus probiotics on HMO compositions after supplementation to pregnant women at threatening preterm delivery: design of the randomized clinical PROMO trial.

BACKGROUND: As an indigestible component of human breast milk, Human Milk Oligosaccharides (HMOs) play an important role as a substrate for the establishing microbiome of the newborn. They have further been shown to have beneficial effects on the immune system, lung and brain development. For preterm infants HMO composition of human breast milk may be of particular relevance since the establishment of a healthy microbiome is challenged by multiple disruptive factors associated with preterm birth, such as cesarean section, hospital environment and perinatal antibiotic exposure. In a previous study it has been proposed that maternal probiotic supplementation during late stages of pregnancy may change the HMO composition in human milk. However, there is currently no study on pregnancies which are threatened to preterm birth. Furthermore, HMO composition has not been investigated in association with clinically relevant outcomes of vulnerable infants including inflammation-mediated diseases such as sepsis, necrotizing enterocolitis (NEC) or chronic lung disease. MAIN BODY: A randomized controlled intervention study (PROMO = probiotics for human milk oligosaccharides) has been designed to analyze changes in HMO composition of human breast milk after supplementation of probiotics (Lactobacillus acidophilus, Bifidobacterium lactis and Bifidobacterium infantis) in pregnancies at risk for preterm birth. The primary endpoint is HMO composition of 3-fucosyllactose and 3'-sialyllactose in expressed breast milk. We estimate that probiotic intervention will increase these two HMO levels by 50% according to the standardized mean difference between treatment and control groups. As secondary outcomes we will measure preterm infants' clinical outcomes (preterm birth, sepsis, weight gain growth, gastrointestinal complications) and effects on microbiome composition in the rectovaginal tract of mothers at delivery and in the gut of term and preterm infants by sequencing at high genomic resolution. Therefore, we will longitudinally collect bio samples in the first 4 weeks after birth as well as in follow-up investigations at 3 months, one year, and five years of age. CONCLUSIONS: We estimate that probiotic intervention will increase these two HMO levels by 50% according to the standardized mean difference between treatment and control groups. The PROMO study will gain insight into the microbiome-HMO interaction at the fetomaternal interface and its consequences for duration of pregnancy and outcome of infants.

First-Day-of-Life Rectal Swabs Fail To Represent Meconial Microbiota Composition and Underestimate the Presence of Antibiotic Resistance Genes.

The human gut microbiome plays a vital role in health and disease. In particular, the first days of life provide a unique window of opportunity for development and establishment of microbial community. Currently, stool samples are known to be the most widely used sampling approach for studying the gut microbiome. However, complicated sample acquisition at certain time points, challenges in transportation, and patient discomfort underline the need for development of alternative sampling approaches. One of the alternatives is rectal swabs, shown to be a reliable proxy for gut microbiome analysis when obtained from adults. Here, we compare the usability of rectal swabs and meconium paired samples collected from infants on the first days of life. Our results indicate that the two sampling approaches display significantly distinct patterns in microbial composition and alpha and beta diversity as well as detection of resistance genes. Moreover, the dissimilarity between the two collection methods was greater than the interindividual variation. Therefore, we conclude that rectal swabs are not a reliable proxy compared to stool samples for gut microbiome analysis when collected on the first days of a newborn's life. IMPORTANCE Currently, there are numerous suggestions on how to ease the notoriously complex and error-prone methodological setups to study the gut microbiota of newborns during the first days of life. Especially, meconium samples are regularly failing to yield meaningful data output and therefore have been suggested to be replaced by rectal swabs as done in adults as well. We find this development toward a simplified method to be producing dramatically erroneous results, skewing data interpretation away from the real aspects to be considered for neonatal health during the first days of life. We have put together our knowledge on this critical aspect with careful consideration and identified the failure of rectal swabs to be a replacement for sampling of meconium in term-born newborns.

Alpha-Particle Exposure Induces Mainly Unstable Complex Chromosome Aberrations which do not Contribute to Radiation-Associated Cytogenetic Risk.

The mechanism underlying the carcinogenic potential of α radiation is not fully understood, considering that cell inactivation (e.g., mitotic cell death) as a main consequence of exposure efficiently counteracts the spreading of heritable DNA damage. The aim of this study is to improve our understanding of the effectiveness of α particles in inducing different types of chromosomal aberrations, to determine the respective values of the relative biological effectiveness (RBE) and to interpret the results with respect to exposure risk. Human peripheral blood lymphocytes (PBLs) from a single donor were exposed ex vivo to doses of 0-6 Gy X rays or 0-2 Gy α particles. Cells were harvested at two different times after irradiation to account for the mitotic delay of heavily damaged cells, which is known to occur after exposure to high-LET radiation (including α particles). Analysis of the kinetics of cells reaching first or second (and higher) mitosis after irradiation and aberration data obtained by the multiplex fluorescence in situ hybridization (mFISH) technique are used to determine of the cytogenetic risk, i.e., the probability for transmissible aberrations in surviving lymphocytes. The analysis shows that the cytogenetic risk after α exposure is lower than after X rays. This indicates that the actually observed higher carcinogenic effect of α radiation is likely to stem from small scale mutations that are induced effectively by high-LET radiation but cannot be resolved by mFISH analysis.

Blood group AB increases risk for surgical necrotizing enterocolitis and focal intestinal perforation in preterm infants with very low birth weight.

Necrotizing enterocolitis (NEC) and focal intestinal perforation (FIP) are two of the most common emergencies of the gastrointestinal tract in preterm infants with very low birth weight (VLBW, birth weight < 1500 g). Identification of risk factors among these children is crucial for earlier diagnosis and prompt intervention. In this study, we investigated a relationship between ABO blood groups and the risk for surgical NEC/FIP. We genotyped the ABO locus (rs8176746 and rs8176719) in VLBW infants enrolled in a prospective, population-based cohort study of the German Neonatal Network (GNN). Of the 10,257 VLBW infants, 441 (4.3%) had surgical NEC/FIP. In univariate analyses, the blood group AB was more prevalent in VLBW infants with surgical NEC/FIP compared to non-AB blood groups (OR 1.51, 95% CI 1.07-2.13, p = 0.017; absolute risk difference 2.01%, 95% CI 0.06-3.96%). The association between blood group AB and surgical NEC/FIP was observed in a multivariable logistic regression model (OR of 1.58, 95% CI 1.10-2.26, p = 0.013) as well. In summary, our study suggests that the risk of surgical NEC and FIP is higher in patients with blood group AB and lower in those having non-AB blood groups.

Gut Dysbiosis With Bacilli Dominance and Accumulation of Fermentation Products Precedes Late-onset Sepsis in Preterm Infants.

BACKGROUND: Gut dysbiosis has been suggested as a major risk factor for the development of late-onset sepsis (LOS), a main cause of mortality and morbidity in preterm infants. We aimed to assess specific signatures of the gut microbiome, including metabolic profiles, in preterm infants <34 weeks of gestation preceding LOS. METHODS: In a single-center cohort, fecal samples from preterm infants were prospectively collected during the period of highest vulnerability for LOS (days 7, 14, and 21 of life). Following 16S rRNA gene profiling, we assessed microbial community function using microbial metabolic network modeling. Data were adjusted for gestational age and use of probiotics. RESULTS: We studied stool samples from 71 preterm infants with LOS and 164 unaffected controls (no LOS/necrotizing enterocolitis). In most cases, the bacteria isolated in diagnostic blood culture corresponded to the genera in the gut microbiome. LOS cases had a decelerated development of microbial diversity. Before onset of disease, LOS cases had specific gut microbiome signatures with higher abundance of Bacilli (specifically coagulase-negative Staphylococci) and a lack of anaerobic bacteria. In silico modeling of bacterial community metabolism suggested accumulation of the fermentation products ethanol and formic acid in LOS cases before the onset of disease. CONCLUSIONS: Intestinal dysbiosis preceding LOS is characterized by an accumulation of Bacilli and their fermentation products and a paucity of anaerobic bacteria. Early microbiome and metabolic patterns may become a valuable biomarker to guide individualized prevention strategies of LOS in highly vulnerable populations.

Granulocytic myeloid-derived suppressor cells (GR-MDSC) accumulate in cord blood of preterm infants and remain elevated during the neonatal period.

Preterm delivery is the leading cause of perinatal morbidity and mortality. Among the most important complications in preterm infants are peri- or postnatal infections. Myeloid-derived suppressor cells (MDSC) are myeloid cells with suppressive activity on other immune cells. Emerging evidence suggests that granulocytic MDSC (GR-MDSC) play a pivotal role in mediating maternal-fetal tolerance. The role of MDSC for postnatal immune-regulation in neonates is incompletely understood. Until the present time, nothing was known about expression of MDSC in preterm infants. In the present pilot study, we quantified GR-MDSC counts in cord blood and peripheral blood of preterm infants born between 23 + 0 and 36 + 6 weeks of gestation (WOG) during the first 3 months of life and analysed the effect of perinatal infections. We show that GR-MDSC are increased in cord blood independent of gestational age and remain elevated in peripheral blood of preterm infants during the neonatal period. After day 28 they drop to nearly adult levels. In case of perinatal or postnatal infection, GR-MDSC accumulate further and correlate with inflammatory markers C-reactive protein (CRP) and white blood cell counts (WBC). Our results point towards a role of GR-MDSC for immune-regulation in preterm infants and render them as a potential target for cell-based therapy of infections in these patients.

Regulatory T cell frequencies are increased in preterm infants with clinical early-onset sepsis.

The predisposition of preterm neonates to invasive infection is, as yet, incompletely understood. Regulatory T cells (Tregs ) are potential candidates for the ontogenetic control of immune activation and tissue damage in preterm infants. It was the aim of our study to characterize lymphocyte subsets and in particular CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) Tregs in peripheral blood of well-phenotyped preterm infants (n = 117; 23 + 0 - 36 + 6 weeks of gestational age) in the first 3 days of life in comparison to term infants and adults. We demonstrated a negative correlation of Treg frequencies and gestational age. Tregs were increased in blood samples of preterm infants compared to term infants and adults. Notably, we found an increased Treg frequency in preterm infants with clinical early-onset sepsis while cause of preterm delivery, e.g. chorioamnionitis, did not affect Treg frequencies. Our data suggest that Tregs apparently play an important role in maintaining maternal-fetal tolerance, which turns into an increased sepsis risk after preterm delivery. Functional analyses are needed in order to elucidate whether Tregs have potential as future target for diagnostics and therapeutics.

Increased risk for bronchitis after discharge in non-vaccinated very low birth weight infants.

BACKGROUND: In very low birth weight (VLBW) infants, obstructive bronchitis is a frequent cause of hospital re-admission. For VLBW infants, early vaccinations starting at 2 months after birth have been recommended. OBJECTIVE: To analyze risk factors for bronchitis during the first year after discharge and the effects of in-hospital standard vaccination (hexavalent/pneumococci) and/or RSV immunoprophylaxis with palivizumab. METHODS: A standardized questionnaire was sent to the parents of VLBW infants 7 month after discharge. The reported episodes of bronchitis were correlated with clinically recorded parameters including risk factors for pulmonary morbidity. The effects of in-hospital vaccination were assessed in a subgroup discharged after day 60. RESULTS: A sample of 1 967 responses of infants born 2009-2011 was analyzed. Risk factors for bronchitis were male gender and older siblings. 24% of the population had episodes of bronchitis. In the subgroup discharged after day 60, episodes of bronchitis were reported for 31% of infants who were not vaccinated in-hospital. A significant reduction of the bronchitis rate was found in infants who received palivizumab±standard vaccination (17% bronchitis, p=0.003). Interestingly, in-hospital standard vaccination without RSV immunoprophylaxis was protective (20% bronchitis; p=0.037) as well. CONCLUSIONS: Non-vaccinated male VLBW infants with older siblings are at increased risk for bronchitis during the first year after discharge. Vaccination according to schedule seems to have protective effects, while underlying mechanisms are unknown. The rate of timely vaccination in preterm infants should be increased.

Clinical phenotype and functional analysis of a rare XIAP/BIRC4 mutation.

X-linked lymphoproliferative syndromes (XLP) are rare primary immunodeficiencies. Mutations within the XIAP/BIRC4 gene characterize XLP type 2 and cause XIAP deficiency. We present the case of a 5-year-old boy with a novel mutation of the XIAP/BIRC4 gene and describe the immunological phenotype for the first time. We characterized the distinct immunological phenotype and evaluated the family history accordingly.

Major contributors to hospital mortality in very-low-birth-weight infants: data of the birth year 2010 cohort of the German Neonatal Network.

The German Neonatal Network (GNN) is a prospective cohort study with the focus on long term development of very-low-birth-weight infants. It was the aim of this study to determine detailed information on causes of mortality in the GNN birth cohort 2010.Major contributors to hospital mortality were recorded by the attending neonatologists for the cohort of very-low-birth-weight (VLBW) infants born in centres of the German Neonatal Network (GNN) in 2010. The data quality was approved by on-site monitoring.2 221 VLBW infants were born in GNN centres in 2010, and death occurred in 221 infants. Male infants carried a higher risk than females (58.8% males among non-survivors vs. 51.7% among survivors, p=0.047). In 11 infants, the major contributor to death was not determined by the attending neonatologist. In 25 infants born at the limit of viability, comfort palliative care was primarily initiated and 14 infants had lethal malformations. The majority of non-survivors suffered from inflammatory diseases including sepsis- or necrotizing enterocolitis (NEC)-associated death (n=56). Respiratory pathology was a major contributor to death in 65 infants including 11 infants who died from pulmonary haemorrhage.Potentially preventable complications of preterm birth such as sepsis, NEC and pulmonary haemorrhage predominate the major contributors to mortality in the GNN 2010 cohort. In order to decrease the rate of these associated deaths, future trials should focus on prophylaxis and therapy optimization strategies for these outcomes.

Galectin-3 in cord blood of term and preterm infants.

In recent years galectin-3 has gained attention as a signalling molecule, mainly in inflammatory diseases. Data on galectin-3 expression in neonates, however, are limited, and expression of this lectin in cord blood has not yet been reported. The aim of this study was to determine galectin-3 levels in cord blood of term and preterm neonates as well as galectin-3 levels in cord blood of term neonates after stimulation with the prevalent pathogen Streptococcus agalactiae. Cord blood samples were incubated for 24 h and galectin-3 levels were assessed by enzyme-linked immunosorbent assay. There is a positive correlation between gestational age and galectin-3 levels in cord blood. Expression of galectin-3 is significantly higher in cord blood of small-for-gestational-age infants compared to appropriate-for-gestational-age infants. Stimulation with an invasive but not with a colonizing strain of S. agalactiae induced expression of galectin-3. Galectin-3 is expressed constitutively in cord blood of neonates and seems to play a role in the innate immunity of this population.

Pandemic A/H1N1(2009) influenza infections in very-low-birth-weight infants--a case series from the German Neonatal Network.

6 cases of clinical influenza A/H1N1(2009) infections were reported within the multi-center German Neonatal Network (GNN) during the primary hospital stay in the pandemic season 2009/2010 and 2010/2011. Clinical symptoms varied from transient hyperthermia to apnea and severe respiratory distress. 1 fatal course with systemic inflammatory response after perinatal transmission of A/H1N1(2009) was observed. Oseltamivir treatment in 3/6 infants was without side effects. The reported cases have major implications for the management of VLBW infants: i) fatal courses after perinatal transmission are possible, ii) postnatal A/H1N1(2009) infection may result in life threatening events at a time when the infant is otherwise stable, iii) vaccination should be recommended for parents and medical staff to avoid nosocomial transmission, iv) more data are needed on the benefit and harm of antiviral drugs in preterm infants, v) neonatologists should suspect A/H1N1(2009) infection when unexplained sepsis-like or respiratory symptoms occur in VLBW infants.

Polymorphisms of interleukin 18 in the genetics of preterm birth and bronchopulmonary dysplasia.

Interleukin 18 (IL-18) is an important cytokine and involved in the pathogenesis and genetics of many diseases. The authors studied two different populations of preterm infants to test whether polymorphisms within IL-18 are in association with prematurity itself or with typical pulmonary disease or measurements seen in preterm infants, such as bronchopulmonary dysplasia, pneumothoraces and application of surfactant, inhalation or mechanical ventilation. Whereas the first population of 228 preterm infants showed strong association of IL-18 with preterm birth (p<0.001), this was not confirmed in the second population of 346 preterm infants. In addition, no association with any lung condition of prematurity was observed. The authors conclude that IL-18 does not play an important role in the genetics of preterm birth nor in the development of bronchopulmonary dysplasia and other lung complications in preterm infants. Caution must be taken in the interpretation of the results of genetic association studies performed in one population.

Surfactant without intubation in preterm infants with respiratory distress: first multi-center data.

BACKGROUND: Recently in a report of a single center a method has been described to apply surfactant via a thin endotracheal catheter to very low birth weight infants spontaneously breathing with nasal continuous positive airway pressure. We now analyzed available multicenter data. PATIENTS AND METHODS: In a multicenter study investigating genetic risk factors, clinical and outcome data and data of antenatal and postnatal treatment of infants with a birth weight below 1,500 g were prospectively recorded. The measures of infants treated with the new method of surfactant application were compared to those of infants who received standard care. The analysis was restricted to infants with a gestational age below 31 weeks (n=1,541). RESULTS: 319 infants were treated with the new method and 1,222 with standard care. The need for mechanical ventilation during the first 72 h (29% vs. 53%, p<0.001), the rate of bronchopulmonary dysplasia defined as oxygen at 36 weeks of postmenstrual age (10.9 % vs. 17.5%, p=0.004) and the rate of death or bronchopulmonary dysplasia were significantly lower in the treatment group than in the standard care group. Surfactant, theophyllin, caffeine and doxapram were significantly more often and analgetics, catecholamines and dexamethasone were significantly less frequently used in the treatment group. CONCLUSIONS: A new method of surfactant application was associated with a lower prevalence of mechanical ventilation and better pulmonary outcome. A prospective controlled trial is required to determine whether this approach is superior to standard care.

Attenuation of monocyte proinflammatory cytokine responses to Neisseria meningitidis in children by erythropoietin.

Meningococcal disease is a leading infectious cause of death in children in industrialized countries. The induction of high levels of proinflammatory cytokines has been implicated in the pathogenesis of Neisseria meningitidis-related multi-organ failure. Here, we demonstrate that N. meningitidis serotypes A and B induce significantly higher levels of tumour necrosis factor (TNF)-alpha positive cells in vitro in infants and young children compared with adults (serotype A/B; infants: 64.9%/63.9%; children: 77.8%/64.3% versus adults: 27.7%/32%; P < 0.005). Serotype A induces also higher levels of interleukin (IL)-6 positive cells in neonates and infants compared with adults (serotype A; newborn 55.4%; infants 58.8% versus adults 49%; P < 0.05). Treatment with human recombinant erythropoietin in vitro resulted in significant attenuation of the N. meningitidis-induced proinflammatory response in all age groups (reduction rate of erythropoietin for IL-6 after stimulation with serotype B: newborn 28%, infants 15%, children 23% and adults 28% and for TNF-alpha after stimulation with serotype B: newborn 27%, infants 22%, children 20% and adults 28%; P < 0.05). We conclude that (i) Neisseria meningitidis induces a higher TNF-alpha response in infants and children compared with adults and (ii) erythropoietin was able to attenuate IL-6 and TNF-alpha production in all investigated age groups. These data may explain the high incidence of meningococcal infection in infants and makes erythropoietin a potentially attractive candidate for interventional strategies in an otherwise devastating course of the disease.

Characterisation of the host inflammatory response to Staphylococcus epidermidis in neonatal whole blood.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens causing late-onset sepsis, and gestational age is the most important risk factor for these infections. OBJECTIVE: To characterise innate immune responses to S epidermidis by assessment of whole blood in vitro cytokine production in a large group of preterm and term infants. RESULTS: The S epidermidis-induced in vitro production of proinflammatory cytokines such as intracytoplasmic interleukin (IL) 6 and tumour necrosis factor alpha in cord blood samples was found to be dependent on gestational age (R = 0.279, 95% CI 0.10 to 0.44, p = 0.002; R = 0.251, 95% CI 0.07 to 0.41, p = 0.005, respectively; n = 121). In contrast, the production of anti-inflammatory cytokines such as IL10 and transforming growth factor beta was not associated with gestational age. When different stimulation strategies were compared, a strong correlation was noted for cytokine responses after lipopolysaccharide and S epidermidis exposure--that is, IL6 (R = 0.431, 95% CI 0.29 to 0.55, p<0.001, n = 161) and IL10 (R = 0.332, 95% CI 0.18 to 0.47, p<0.001, n = 161). In addition, a lower IL6 production was found in supernatants of whole blood cultures infected with a clinically isolated IcaABD-positive (biofilm production) strain compared with a control IcaABD-negative ATCC strain (p = 0.009). CONCLUSIONS: These in vitro data suggest that proinflammatory responses to S epidermidis are dependent on gestational age in preterm infants, whereas the counteracting anti-inflammatory response to S epidermidis may not be directly related to gestational age. Individual host factors may have a role as well as bacterial determinants, such as biofilm production. Further studies are encouraged to investigate the different aspects of innate immune responses to CoNS in vivo.

Predictive value of neurodevelopmental assessment versus evaluation of general movements for motor outcome in preterm infants with birth weights <1500 g.

PURPOSE: The aim of this study was to make a comparison of predictive values of neurodevelopmental assessment and evaluation of videotaped spontaneous movements of premature infants for motor outcome. METHODS: We performed a prospective longitudinal study of 103 VLBW infants, 96 (455-1490 g, 24-35 weeks gestational age) including (a) a neurodevelopmental assessment based on criteria by Amiel-Tison/Grenier at 40 weeks postconceptional age, 3 and 20 months corrected age; (b) an evaluation of general movements with fidgety character, based on criteria by Prechtl, at 3 months; and (c) a standardized testing using the Griffiths Developmental Motor Scale at 20 months. We calculated sensitivity, specificity and predictive values for each method. RESULTS: For predicting motor outcome, the assessment of general movements (GM) had a positive predictive value of 89% and negative predictive value of 84%; neurodevelopmental assessment (NA) at 40 weeks had a positive predictive value of 33% and negative predictive value of 88%, respectively, with similar results for neurodevelopmental assessment at age 3 months. CONCLUSIONS: Normal motor outcome of VLBW infants may be accurately predicted by clinical neurodevelopmental assessment, but for adverse outcomes, evaluation of general movements (fidgety movements) is superior. GM assessment has a high predictive value, especially for CP, but it needs to be complemented by NA for non-CP outcomes. It is a simple, repeatable and non-intrusive technique, and may be a valuable method for the early detection of central nervous system impairment in VLBW infants in routine follow-up.

Reduced IL-10 production and -receptor expression in neonatal T lymphocytes.

AIM: To further evaluate the underlying mechanism of a formerly demonstrated immature anti-inflammatory response in neonates (1). METHODS: Interleukin (IL)-10 production was measured by enzyme-linked immunosorbent-assay (ELISA) after anti-CD3/anti-CD28 costimulation of neonatal and adult T cells. IL-10 receptor expression on T lymphocytes, B lymphocytes and monocytes were analysed by flow cytometry in neonates and adult controls. RESULTS: After anti-CD3/anti-CD28 costimulation, IL-10 production of neonatal T lymphocytes was profoundly reduced (median 247 pg/mL vs. 1062 pg/mL, p < 0.0001). IL-10 receptor expression was diminished on neonatal T lymphocytes compared to adults (3% vs. 39.5% IL-10 receptor positive lymphocytes; p < 0.0001). On neonatal B lymphocytes and monocytes the IL-10 receptor expression was comparable to adult controls. CONCLUSION: The strongly reduced IL-10 receptor expression on the main immune regulative T lymphocytes in conjunction with a significantly impaired synthesis of IL-10 may play a crucial role in the formerly demonstrated deficient anti-inflammatory immune response in neonates.

Renal complications associated with human parvovirus B19 infection in early childhood.

A previously healthy two-year-old girl presented with proteinuria and macroscopic haematuria. Laboratory findings included haemolytic anaemia with thrombocytopenia. Interestingly, continuing reticulocytopenia was noted. Therefore an acute parvovirus B19 infection was suspected, which could be confirmed by serological and molecularbiological evidence. This case report underlines renal complications of parvovirus B19 infection in early childhood including haemolytic-uraemic syndrome (HUS)-like episodes, and potential pathogenetic mechanisms are discussed.