Objective To assess the utility of clinical predictors of persistent respiratory morbidity in extremely low gestational age newborns (ELGAN). Study Design We enrolled ELGAN (<29 weeks’ gestation) at ≤7 postnatal days and collected antenatal and neonatal clinical data through 36 weeks’ post-menstrual age. We surveyed caregivers at 3, 6, 9 and 12 months corrected age to identify post-discharge respiratory morbidity, defined as hospitalization, home support (oxygen, tracheotomy, ventilation), medications, or symptoms (cough/wheeze). Infants were classified as post-prematurity respiratory disease (PRD, the primary study outcome), if respiratory morbidity persisted over ≥2 questionnaires. Infants were classified with severe respiratory morbidity if there were multiple hospitalizations, exposure to systemic steroids or pulmonary vasodilators, home oxygen after 3 months or mechanical ventilation, or symptoms despite inhaled corticosteroids. Mixed effects models generated with data available at one day (perinatal) and 36 weeks’ postmenstrual age were assessed for predictive accuracy. Results Of 724 infants (918±234g, 26.7±1.4 weeks’ gestational age) classified for the primary outcome, 68.6% had PRD; 245/704 (34.8%) were classified as severe. Male sex, intrauterine growth restriction, maternal smoking, race/ethnicity, intubation at birth, and public insurance were retained in perinatal and 36-week models for both PRD and respiratory morbidity severity. The perinatal model accurately predicted PRD (c-statistic 0.858). Neither the 36-week model nor the addition of bronchopulmonary dysplasia (BPD) to the perinatal model improved accuracy (0.856, 0.860); c-statistic for BPD-alone was 0.907. Conclusion Both BPD and perinatal clinical data accurately identify ELGAN at risk for persistent and severe respiratory morbidity at one year. Trial registration ClinicalTrials.gov: NCT01435187

Previous studies have shown reduced hypothalamo-pituitary-adrenal responses to both acute and chronic restraint stressors in rats allowed to ingest highly palatable foods (32% sucrose +/- lard) prior to restraint. In this study we tested the effects of prior access (7 d) to chow-only, sucrose/chow, lard/chow, or sucrose/lard/chow diets on central corticotropin-releasing factor (CRF) expression in rats studied in two experiments, 15 and 240 min after onset of restraint. Fat depot, particularly intraabdominal fat, weights were increased by prior access to palatable food, and circulating leptin concentrations were elevated in all groups. Metabolite concentrations were appropriate for values obtained after stressors. For unknown reasons, the 15-min experiment did not replicate previous results. In the 240-min experiment, ACTH and corticosterone responses were inhibited, as previously, and CRF mRNA in the hypothalamus and oval nucleus of the bed nuclei of the stria terminalis were reduced by palatable foods, suggesting strongly that both neuroendocrine and autonomic outflows are decreased by increased caloric deposition and palatable food. In the central nucleus of the amygdala, CRF was increased in the sucrose-drinking group and decreased in the sucrose/lard group, suggesting that the consequence of ingestion of sucrose uses different neural networks from the ingestion of lard. The results suggest strongly that ingestion of highly palatable foods reduces activity in the central stress response network, perhaps reducing the feeling of stressors.

Objectives To assess whether late surfactant treatment of extremely low gestational age newborn (ELGAN) infants requiring ventilation at 7–14 days, who often have surfactant deficiency and dysfunction, safely improves survival without bronchopulmonary dysplasia (BPD). Study design ELGAN infants (≤ 28 0/7 weeks) who required mechanical ventilation at 7–14 days were enrolled in a randomized, masked controlled trial at 25 US centers. All infants received inhaled nitric oxide (INO) and either surfactant (calfactant/Infasurf®) or sham instillation every 1–3 days to a maximum of 5 doses while intubated. The primary outcome was survival at 36 weeks postmenstrual age (PMA) without BPD, evaluated by physiologic oxygen/flow reduction. Results Between January 2010 and September 2013, 511 infants were enrolled. There were no differences between treatment groups in mean birth weight (701±164 g), gestational age (25.2±1.2 weeks), percentage <26 weeks (70.6%), race, sex, severity of lung disease at enrollment, or co-morbidities of prematurity. Survival without BPD was not different between treated vs. controls at 36 weeks PMA (31.3% vs. 31.7%; relative benefit 0.98 (95% CI: 0.75, 1.28 p=0.89) or 40 weeks (58.7% vs. 54.1%; relative benefit 1.08:0.92, 1.27 p=0.33). There were no differences between groups in serious adverse events, co-morbidities of prematurity, nor in the severity of lung disease to 36 weeks. Conclusions Late treatment with up to 5 doses of surfactant in ventilated premature infants receiving iNO was well tolerated but did not improve survival without BPD at 36 or 40 weeks. Pulmonary and neurodevelopmental assessments are ongoing.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

Corticosterone (B) increases and insulin decreases food intake. However, in streptozotocin (STZ)-diabetic rats with high B, low insulin replacement promotes lard intake. To test the role of the liver on this, rats were given STZ and infused with insulin or vehicle into either the superior mesenteric or right jugular vein. Controls were nondiabetic; all rats were treated with high B. After 5 d, all rats were offered lard, 32% sucrose, chow, and water ad libitum until d 10. Diabetes exacerbated body weight loss from high B; this was prevented by insulin into the jugular, but not superior mesenteric, vein. Without insulin, STZ groups essentially consumed only chow; controls increased caloric intake about equally from the three sources. Insulin into both sites reduced chow and increased lard intake. Although circulating insulin was increased only by jugular infusion, plasma glucose and liver glycogen were similar after insulin into both sites. Fat depot weights differed: sc fat was heavier after jugular and mesenteric fat was heavier after mesenteric insulin infusions. We conclude that there are important site-specific effects of insulin in regulating the choice of, but not total, caloric intake, body weight, and fat storage in diabetic rats with high B. Furthermore, lard intake might be regulated by an insulin-derived, liver-mediated signal because superior mesenteric insulin infusion had similar effects on lard intake to jugular infusion but did not result in elevated circulating insulin levels likely associated with liver insulin removal.