Gut Sphingolipid Composition as a Prelude to Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) remains a major challenge in neonatology. Little is known about NEC pathophysiology apart from the presence of pre-event gut dysbiosis. Here, we applied broad range metabolomics to stools obtained 1-5 days before NEC developed from 9 cases (9 samples) and 19 (32 samples) controls matched for gestational age at birth and birth weight. The 764 identified metabolites identified six pathways that differ between cases and controls. We pursued sphingolipid metabolism because cases had decreased ceramides and increased sphingomyelins compared to controls, and because of the relevance of sphingolipids to human inflammatory disorders. Targeted analysis of samples from 23 cases and 46 controls confirmed the initial broad range observations. While metabolites provided only 73% accuracy of classification by machine learning, hierarchical clustering defined a sphingolipid associated grouping that contained 60% of the cases but only 13% of the controls, possibly identifying a pathophysiologically distinct subset of NEC. The clustering did not associate with any of the analyzed clinical and sample variables. We conclude that there are significant changes in sphingolipid metabolism components in pre-NEC stools compared to controls, but our data urge circumspection before using sphingolipids as broadly applicable predictive biomarkers.

Emergence of community-associated methicillin-resistant Staphylococcus aureus strains in the neonatal intensive care unit: an infection prevention and patient safety challenge.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause significant morbidity and mortality in neonatal intensive care units (NICUs). We characterized the clinical and molecular epidemiology of MRSA strains colonizing NICU patients. Nasal MRSA isolates (n = 250, from 96 NICU patients) recovered through active surveillance from 2009 to 2014 were characterized with staphylococcal cassette chromosome mec (SCCmec) typing and detection of mupA (marker of high-level mupirocin resistance) and qacA/B (marker associated with chlorhexidine resistance). Factors associated with community-associated (CA-) or healthcare-associated (HA-) MRSA were evaluated. The overall prevalence of MRSA nasal colonization was 3.9%. Of 96 neonates in our retrospective cohort, 60 (63%) were colonized with CA-MRSA strains and 35 (36%) were colonized with HA-MRSA strains. Patients colonized with HA-MRSA were more likely to develop MRSA infections than patients colonized with CA-MRSA (13/35, 37% versus 8/60, 13%; p 0.007), although the interval from colonization to infection was shorter in CA-MRSA-colonized infants (median 0 days, range -1 to 4 versus HA-MRSA-colonized infants, 7 days, -1 to 43; p 0.005). Maternal peripartum antibiotics were associated with CA-MRSA colonization (adjusted odds ratio (aOR) 8.7; 95% CI 1.7-45.0); intubation and surgical procedures were associated with HA-MRSA colonization (aOR 7.8; 95% CI 1.3-47.6 and aOR 6.0; 95% CI 1.4-24.4, respectively). Mupirocin- and chlorhexidine-resistant MRSA was isolated from four and eight patients, respectively; carriage of a mupirocin-resistant strain precluded decolonization. CA-MRSA strains are prominent in the NICU and associated with distinct risk factors. Given community reservoirs for MRSA acquisition and transmission, novel infection prevention strategies are needed.

Small bowel resection induces long-term changes in the enteric microbiota of mice.

PURPOSE: The enteric microbiome is known to play a major role in healthy gut homeostasis and several disease states. It may also contribute to both the intestinal recovery and complications that occur in patients with short bowel syndrome. The extent and nature of alterations to the gut microbiota following intestinal resection, however, are not well studied in a controlled setting. The purpose of this investigation is to characterize the effects of massive small bowel resection on the murine enteric microflora. METHODS: Wild-type C57BL6 mice, following a week of acclamation to a liquid rodent diet, underwent either 50% proximal small bowel resection (SBR) or a sham operation. Mice were sacrificed, and enteric contents from the small bowel, cecum, and stool were harvested at 7 and 90 days post-operatively. DNA was isolated, and the V3-V5 regions of the 16s rRNA gene amplified and pyrosequenced on a Roche 454 platform. Sequences were clustered into operation taxonomic units and classified. Communities were then analyzed for diversity and phylogenic composition. RESULTS: In the long-term group, the microbes inhabiting the ileum of mice undergoing SBR and sham operation differed significantly at the genus level (p < 0.001). Small bowel contents collected before and after SBR also differed significantly (p = 0.006). This was driven by an increase in Lactobacillus and decrease in Enterobacteriaceae species in mice undergoing SBR. No difference was seen in the long-term stool or in stool, cecal, or ileal contents in the short-term. No difference in microbial community diversity was found in any group. CONCLUSION: Bowel resection induces long-term changes in the microbial community of the murine ileum, but not at more distal sites of the gastrointestinal tract. The increase in Lactobacillus encountered small bowel of resected mice correlates with limited previous studies. These changes may reflect an adaptive response of the microbiota to maximize energy extraction, but further studies are needed to establish the role played by this altered community.

The age of necrotizing enterocolitis onset: an application of Sartwell's incubation period model.

OBJECTIVE: Model age of necrotizing enterocolitis (NEC) onset applying Sartwell's model of incubation periods, and examine its relationship to gestational age (GA). STUDY DESIGN: Retrospective chart review of St Louis Children's Hospital neonates diagnosed with NEC (≥Bell's stage II) from 2004 to 2008, inclusive. RESULT: The relationship between age of NEC (N=84 cases) onset and GA best fits a non-linear model, with infants ≤28 weeks having a disproportionately longer time to onset than older GA groups and explained 50.3% of the variability in age of NEC onset. Additional clinical variables provided no improvement in explaining age of NEC onset. Application of Sartwell's model to age of NEC onset proved a good fit, when birth is used as the common exposure episode, and age is equivalent of the incubation period. CONCLUSION: The relationship between day of NEC diagnosis and GA is non-linear, with lower GA infants having disproportionately longer time to onset. Despite these GA differences, the fit to Sartwell's model for incubation periods model is consistent with NEC being a consequence of an event that occurs at or soon after birth.

Multiple births and outcome.

The rate of multiple-gestation pregnancies has grown exponentially over the last few decades and is responsible for the steady increase in the birth rate of low-birth weight infants. As a group, infants of multiple-gestation pregnancies have higher mortality and morbidity than singleton pregnancies. The increase in adverse outcomes is related directly to the increased risk for preterm delivery and low-birth weight, and not to the multiple gestation itself. Outcomes for multiple-gestation infants appear to be similar whether conceived spontaneously or through artificial reproductive technology. Efforts to reduce the birth rate of low-birth weight infants should target multiple-gestation pregnancies.

Functional and pathological effects of prolonged hyperoxia in neonatal mice.

Bronchopulmonary dysplasia (BPD) commonly develops in premature infants. An improved understanding of the pathophysiology of BPD requires better models. In this study, neonatal FVB/N mice were exposed to room air or 85% oxygen for 28 days. Neonatal hyperoxia resulted in decreased alveolar septation, increased terminal air space size, and increased lung fibrosis. These changes were evident after 7 days and more pronounced by 28 days. Decreased alveolarization was preceded by decreased proliferation of lung cells. After 3 days of hyperoxia, cell proliferation was decreased compared with room air littermates. Cell proliferation continued to be decreased in the first 2 wk but normalized by 4 wk. Hyperoxia caused an increased number of inflammatory cells in lung tissue and in lung lavage fluid. Analysis of lung tissue RNA by RT-PCR showed that hyperoxia increased expression of the proinflammatory cytokines interleukin-1alpha and macrophage inflammatory protein-1alpha. Prolonged neonatal hyperoxia caused functional changes, decreasing lung volume and pulmonary compliance. We conclude that prolonged exposure of neonatal mice to hyperoxia creates a lesion that is very similar to human BPD and suggests that altered cell proliferation may be important in the pathogenesis of chronic neonatal lung disease.

Redox regulation of manganese superoxide dismutase.

The importance of reactive oxygen species (ROS) or changes in cellular redox state in signal transduction and gene regulation is becoming increasingly evident. In this study, we tested the hypothesis that ROS are directly involved in the induction of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) and mediate the induction of MnSOD by tumor necrosis factor-alpha (TNF-alpha). Pretreatment of human pulmonary adenocarcinoma cells H441 with the antioxidants N-acetyl-L-cysteine (NAC) and nordihydroguaiaretic acid (NDGA) blocked MnSOD induction by TNF-alpha, implicating ROS as a signaling agent in this pathway. Treatment of H441 cells with the exogenous oxidants hydrogen peroxide (H2O2) and diamide increased MnSOD mRNA, supporting the hypothesis that ROS directly affect expression of MnSOD. The temporal pattern of MnSOD induction differed for TNF-alpha and H2O2, suggesting distinct signaling pathways. DNA binding of two redox-sensitive transcription factors, NF-kappa B and activator protein (AP)-1, was evaluated. TNF-alpha increased nuclear factor (NF)-kappa B-DNA binding, an effect blocked by pretreatment with NAC. H2O2 did not alter NF-kappa B-DNA binding. There was no evidence of AP-1 binding in cells treated with either TNF-alpha or H2O2. We conclude that ROS directly alter MnSOD expression and are involved in the induction of MnSOD by TNF-alpha.

Human Mn-superoxide dismutase in pulmonary epithelial cells of transgenic mice confers protection from oxygen injury.

To test directly whether mitochondrial Mn-superoxide dismutase (Mn-SOD) protects the lung epithelium from oxygen-induced injury, transgenic mice were produced in which the expression of human Mn-SOD mRNA was directly by transcriptional elements from the human pulmonary surfactant protein C gene. Human Mn-SOD mRNA was expressed in a lung-specific manner, and increased Mn-SOD protein was detected within mitochondria of alveolar Type II and nonciliated bronchiolar cells of the distal respiratory epithelium of the transgenic mice. The activity of Mn-SOD, but not catalase, CuZn-SOD, or glutathione peroxidase, was increased in lungs of transgenic mice. Transgenic mice were highly protected from lung injury during exposure to 95% oxygen, surviving significantly longer than nontransgenic littermates. Pulmonary pathology demonstrated decreased hemorrhage, hyaline membrane formation, and alveolar and interstitial edema in transgenic animals. The finding that increased Mn-SOD in distal respiratory epithelial cells confers protection from oxygen injury provides a basis for novel therapies to protect lung from injury during oxygen therapy of acute and chronic lung diseases.

Tumor necrosis factor-alpha increases Mn-SOD expression: protection against oxidant injury.

Antioxidant enzymes, including superoxide dismutase, are important for protecting the lung against O2 injury. Manganese superoxide dismutase (Mn-SOD) is a superoxide anion (O2-.) scavenger located in the mitochondria, a primary site of O2-. production during hyperoxia. We studied the effects of tumor necrosis factor (TNF-alpha), a macrophage-derived cytokine, on Mn-SOD expression in human pulmonary adenocarcinoma cells. TNF-alpha significantly increased Mn-SOD activity and mRNA in a dose-and time-dependent manner. Mn-SOD activity was increased 3-fold and mRNA 20-fold after a 48-h incubation with TNF-alpha (25 ng/ml). To examine the mechanism of this increase, cells were incubated for 48 h with TNF-alpha (25 ng/ml) with or without cycloheximide (10 microns) or actinomycin D (10 micrograms/ml). Actinomycin D blocked the induction of Mn-SOD mRNA by TNF-alpha, but cycloheximide did not. These findings suggest that the effect of TNF-alpha requires gene transcription but not synthesis of new protein intermediates. To test the hypothesis that increased Mn-SOD protects against oxidative injury, pulmonary adenocarcinoma cells were incubated in TNF-alpha (25 ng/ml) for 48 h and then exposed to paraquat (PQ+), an intracellular O2-. generator. Cells pretreated with TNF-alpha had significantly improved survival in PQ+ compared with controls. At the LD50 (6 microns) for control cells, 95% of TNF-alpha-treated cells survived, 85% at the LD75 (10 microns), and 77% at the LD90 (14 microns). Our results suggest that the induction of Mn-SOD by TNF-alpha in pulmonary adenocarcinoma cells is pretranslationally mediated and that increasing Mn-SOD activity with TNF-alpha confers protection against O2 radicals.

Tumor necrosis factor-alpha inhibits expression of pulmonary surfactant protein.

Tumor necrosis factor-alpha (TNF-alpha) decreased the expression of pulmonary surfactant proteins SP-A and SP-B in human pulmonary adenocarcinoma cell lines. The effect of TNF alpha on SP-A content and mRNA in the pulmonary adenocarcinoma cell line, H441-4, was concentration and time dependent. TNF alpha decreased the cellular content of SP-A to less than 10% of control 48 h after addition. TNF alpha decreased de novo synthesis of SP-A and decreased the accumulation of SP-A in media. SP-A mRNA was decreased within 12 h of addition of TNF alpha, with nearly complete loss of SP-A mRNA observed after 24 h. Inhibitory effects of TNF alpha on SP-A mRNA were dose-related with nearly complete inhibition of SP-A mRNA caused by 25 ng/ml TNF alpha. The effects of TNF alpha on SP-A were distinct from the effects of interferon gamma which increased SP-A content approximately twofold in H441-4 cells. TNF alpha also decreased the content of SP-B mRNA. In contrast to the inhibitory effect of TNF alpha on SP-A and SP-B mRNA, TNF alpha increased mRNA encoding human manganese superoxide dismutase (Mn-SOD). TNF alpha did not inhibit growth, alter cell viability or beta-actin mRNA in either cell line. These in vitro studies demonstrate the marked pretranslational inhibitory effects of the cytokine, TNF alpha, on the expression of pulmonary surfactant proteins, SP-A and SP-B. The results support the concept that macrophage-derived cytokines may control surfactant protein expression.