Angel A. Luciano

Magnesium Decreases Inflammatory Cytokine Production: A Novel Innate Immunomodulatory Mechanism

MgSO4 exposure before preterm birth is neuroprotective, reducing the risk of cerebral palsy and major motor dysfunction. Neonatal inflammatory cytokine levels correlate with neurologic outcome, leading us to assess the effect of MgSO4 on cytokine production in humans. We found reduced maternal TNF-α and IL-6 production following in vivo MgSO4 treatment. Short-term exposure to a clinically effective MgSO4 concentration in vitro substantially reduced the frequency of neonatal monocytes producing TNF-α and IL-6 under constitutive and TLR-stimulated conditions, decreasing cytokine gene and protein expression, without influencing cell viability or phagocytic function. In summary, MgSO4 reduced cytokine production in intrapartum women, term and preterm neonates, demonstrating effectiveness in those at risk for inflammation-associated adverse perinatal outcomes. By probing the mechanism of decreased cytokine production, we found that the immunomodulatory effect was mediated by magnesium and not the sulfate moiety, and it was reversible. Cellular magnesium content increased rapidly upon MgSO4 exposure, and reduced cytokine production occurred following stimulation with different TLR ligands as well as when magnesium was added after TLR stimulation, strongly suggesting that magnesium acts intracellularly. Magnesium increased basal IĸBα levels, and upon TLR stimulation was associated with reduced NF-κB activation and nuclear localization. These findings establish a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis.

Development of the preterm infant gut microbiome: a research priority

The very low birth weight (VLBW) infant is at great risk for marked dysbiosis of the gut microbiome due to multiple factors, including physiological immaturity and prenatal/postnatal influences that disrupt the development of a normal gut flora. However, little is known about the developmental succession of the microbiota in preterm infants as they grow and mature. This review provides a synthesis of our understanding of the normal development of the infant gut microbiome and contrasts this with dysbiotic development in the VLBW infant. The role of human milk in normal gut microbial development is emphasized, along with the role of the gut microbiome in immune development and gastroenteric health. Current research provides evidence that the gut microbiome interacts extensively with many physiological systems and metabolic processes in the developing infant. However, to the best of our knowledge, there are currently no studies prospectively mapping the gut microbiome of VLBW infants through early childhood. This knowledge gap must be filled to inform a healthcare system that can provide for the growth, health, and development of VLBW infants. The paper concludes with speculation about how the VLBW infants’ gut microbiome might function through host-microbe interactions to contribute to the sequelae of preterm birth, including its influence on growth, development, and general health of the infant host.

Preterm Labor and Chorioamnionitis Are Associated with Neonatal T Cell Activation

Background Preterm parturition is characterized by innate immune activation and increased proinflammatory cytokine levels. This well established association leads us to hypothesize that preterm delivery is also associated with neonatal T lymphocyte activation and maturation. Methodology/Principal Findings Cord blood samples were obtained following term, preterm, and deliveries complicated by clinical chorioamnionitis. Activation marker expression was quantitated by flow cytometric analysis. Infants born following preterm delivery demonstrated enhanced CD4+ T lymphocyte activation, as determined by CD25 (Term 9.72% vs. Preterm 17.67%, p = 0.0001), HLA-DR (Term 0.91% vs. Preterm 1.92%, p = 0.0012), and CD69 expression (Term 0.38% vs. Preterm 1.20%, p = 0.0003). Neonates delivered following clinical chorioamnionitis also demonstrated increased T cell activation. Preterm neonates had an increased frequency of CD45RO+ T cells. Conclusion/Significance Preterm parturition is associated with neonatal CD4+ T cell activation, and an increased frequency of CD45RO+ T cells. These findings support the concept that activation of the fetal adaptive immune system in utero is closely associated with preterm labor.

Alterations in Regulatory T Cell Subpopulations Seen in Preterm Infants

Regulatory T cells are a population of CD4+ T cells that play a critical role in peripheral tolerance and control of immune responses to pathogens. The purpose of this study was to measure the percentages of two different regulatory T cells subpopulations, identified by the presence or absence of CD31 (Recent thymic emigrants and peripherally induced naïve regulatory T cells), in term and preterm infant cord blood. We report the association of prenatal factors, intrauterine exposure to lipopolysaccharide and inflammation and the percentages of these regulatory T cell subpopulations in term and preterm infants. Cord blood samples were collected from both term and preterm infants and mononuclear cells isolated over a Ficoll-Hypaque cushion. Cells were then stained with fluorochrome-labeled antibodies to characterize regulatory T cell populations and analyzed with multi-color flow cytometry. Cord blood plasma C-reactive protein, and lipopolysaccharide were also measured. Placental pathology was also examined. We report a gestational age-dependent difference in the percentage of total regulatory T cells, in which preterm infants of lower gestational ages have an increased percentage of regulatory T cells. We report the presence of two populations of regulatory T cells (CD31+ and CD31-) in cord blood of term and preterm infants and their association with different maternal and fetal characteristics. Factors associated with differences in the percentage of CD31- Tregs included the use of prenatal antibiotics, steroids and magnesium sulfate. In addition, the percentage of CD31- Tregs was significantly higher in cord blood of preterm pregnancies associated with inflammation and prenatal lipopolysaccharide exposure. The peripheral Treg pool of preterm infants could be altered by prenatal exposure to inflammation and chorioamnionitis; however, the clinical implications of this finding are not yet understood.

Neonatal T-cell maturation and homing receptor responses to Toll-like receptor ligands differ from those of adult naive T cells: relationship to prematurity

Introduction:Inflammation and infection are associated with premature birth and with activation of the fetal immune system. We hypothesized that exposure to microbial Toll-like receptor (TLR) ligands plays an important role in neonatal T-cell maturation and that early exposure to microbial products may result in early T-cell maturation and a tendency for these matured effector cells to change their homing receptor patterns.Results:Expression of the CD45RO marker was induced in term neonatal T cells after in vitro exposure to TLR ligands for 7 days. Interestingly, naive T cells from adult blood were unaffected by TLR ligand exposure. In addition, neonatal T cells had more cells with decreased expression of the α4β7 integrins and increased expression of CCR4 after in vitro exposure of TLR ligands—similar to the expression of these molecules in adult naive T cells.Discussion:These findings are relevant for the understanding of neonatal T-cell maturation and may contribute to our understanding of multiorgan inflammatory complications of prematurity.Methods:Cord blood was obtained from term and preterm infants. Using flow cytometry, we identified a mature (CD45RO+) phenotype in preterm infant cord blood (CB) T cells that had decreased expression of the α4β7 integrins and increased expression of the C-C chemokine receptor 4 (CCR4) as compared with term infant CB.

Depth of bacterial invasion in resected intestinal tissue predicts mortality in surgical necrotizing enterocolitis.

Objective:Up to a third of all infants who develop necrotizing enterocolitis (NEC) require surgical resection of necrotic bowel. We hypothesized that the histopathological findings in surgically resected bowel can predict the clinical outcome of these infants.Study Design:We reviewed the medical records and archived pathology specimens from all patients who underwent bowel resection/autopsy for NEC at a regional referral center over a 10-year period. Pathology specimens were graded for the depth and severity of necrosis, inflammation, bacteria invasion and pneumatosis, and histopathological findings were correlated with clinical outcomes.Result:We performed clinico-pathological analysis on 33 infants with confirmed NEC, of which 18 (54.5%) died. Depth of bacterial invasion in resected intestinal tissue predicted death from NEC (odds ratio 5.39 per unit change in the depth of bacterial invasion, 95% confidence interval 1.33 to 21.73). The presence of transmural necrosis and bacteria in the surgical margins of resected bowel was also associated with increased mortality.Conclusion:Depth of bacterial invasion in resected intestinal tissue predicts mortality in surgical NEC.

Red blood cell transfusions increase fecal calprotectin levels in premature infants.

Objective:We hypothesized that red blood cell (RBC) transfusions influence intestinal inflammation in very low birth weight (VLBW) infants. We also suspected that hematocrit (Hct) at transfusions and RBC storage time correlate with intestinal inflammation.Study Design:VLBW infants, without major congenital defects, intestinal perforation or necrotizing enterocolitis, were enrolled prospectively. Fecal calprotectin (FC) levels were measured from stool samples collected before and after RBC transfusions. Data on Hct and RBC storage time were collected.Result:Data from 42 RBC transfusions given to 26 infants revealed that FC levels increased faster than baseline after RBC transfusions (P=0.018) and were higher in multiple-transfused infants (0 to 48 and >48 h post transfusion, P=0.007 and P=0.005, respectively). Lower Hct and RBC storage >21 days correlated with higher FC levels (P=0.044 and P=0.013, respectively).Conclusion:RBC transfusions, anemia and prolonged RBC storage were associated with an increase in intestinal inflammation.

Frequencies of FoxP3+ naïve T cells are related to both viral load and naïve T cell proliferation responses in HIV disease

HIV infection results in depletion and dysfunction of naïve CD4+ T cells. The mechanisms underlying these deficiencies are not understood. We investigated the frequencies of CD4+ naïve subsets in HIV disease as defined by expression of CD25 and/or FoxP3 and the relationship of these frequencies to naïve T cell proliferation function. We observed increased proportions of CD25+FoxP3+ and CD25+FoxP3– cells and decreased proportions of CD25–FoxP3– cells within the naïve CD4+ cell compartment from HIV‐infected persons compared with findings in healthy donors. These perturbations were related to higher plasma HIV RNA levels but not with higher immune activation, as measured by the proportions of CD38+ memory CD4+ T cells. Naïve T cell proliferation responses to mitogen stimulation were inversely related to the frequencies and absolute numbers of FoxP3+ naïve T cells. MDA, a marker of oxidative stress, and sCD14, a marker of monocyte activation and a surrogate for microbial translocation, were increased in serum samples from HIV+ donors; however, neither marker was related to naïve T cell function in HIV+ donors. These observations suggest that alterations in naïve T cell subset frequencies could contribute to naïve T cell dysfunction in HIV disease, but these alterations are not necessarily the result of chronic immune activation.

Oxygen requirement as a screening tool for the detection of late pulmonary hypertension in extremely low birth weight infants.

BACKGROUND Many extremely low birth weight infants develop pulmonary hypertension late in their clinical course, and over 60% go undetected by early screening echocardiography. At present, no standardised screening protocol exists for detecting late pulmonary hypertension in extremely low birth weight infants. We assessed the utility of oxygen supplementation as a predictor of late pulmonary hypertension. METHODS A retrospective single-centre review of extremely low birth weight infants with no evidence of CHD and those surviving for >30 days was performed. The association between oxygen ⩾30% at day of life 30 and diagnosis of late pulmonary hypertension was estimated with an odds ratio and 95% confidence interval using logistic regression. Doppler echocardiography was used to diagnose pulmonary hypertension in the infants. RESULTS A total of 230 infants met the study criteria. The incidence of late pulmonary hypertension was 8.3% (19/230). Infants with late pulmonary hypertension were more likely to have a lower mean birth weight (667.1±144 versus 799.3±140 g, p=0.001) and more likely to be small for gestational age (47.4 versus 14.2%, p=0.004). Oxygen requirement ⩾30% at day of life 30 was associated with increased risk of late pulmonary hypertension (odds ratio=3.77, 95% confidence interval=1.42-10.00, p=0.008) in univariate analysis and after adjusting for birth weight (odds ratio=2.47, 95% confidence interval=0.89-6.84, p=0.08). CONCLUSIONS The need of oxygen supplementation ⩾30% at day of life 30 may be a good screening tool for detecting late pulmonary hypertension in extremely low birth weight infants.