Faculty Bibliography

PROBLEM/OBJECTIVE:Placental infection induces increased levels of pro-inflammatory cytokines, which have been implicated in the pathogenesis of preterm labor. Endotoxin tolerance is a phenomenon in which exposure to a dose of endotoxin makes tissue less responsive to subsequent exposures. The objective of our study is to determine if repeated exposure to endotoxin will induce a tolerant phenotype in normal human second trimester placental tissue. METHODS OF STUDY/METHODS:Human second trimester placental explants from elective termination of pregnancy were cultured and exposed to endotoxin (LPS). After 24 hours, the media was collected for analysis, and the explants were re-exposed to LPS after adding fresh media for another 24 hours. This process was repeated for a total of 4 LPS doses. The media was collected from each day and analyzed for cytokine levels. RESULTS:The first LPS treatment stimulated the secretion of the pro-inflammatory cytokines IL-1β, TNF-α. However, their production was significantly diminished with repeated LPS doses. Production of anti-inflammatory cytokines, IL-1ra and IL-10, was also stimulated by the first LPS treatment, but secretion was more gradually and moderately decreased with repeated LPS doses compared to the pro-inflammatory cytokines. The ratios of the anti-inflammatory/pro-inflammatory mediators (IL-1ra/IL-1β and IL-10/ TNF-α) indicate a progressively more anti-inflammatory milieu with repeated LPS doses. CONCLUSIONS:Repeated LPS exposure of human second trimester placental tissues induced endotoxin tolerance. We speculate that endotoxin tolerance at the maternal-fetal interface will protect the fetus from exaggerated inflammatory responses after repeated infectious exposure.

Introduction/UNASSIGNED:Premature babies are at increased risk of hypothermia, core body temperature <97°F. Delivery room environment may contribute and lead to complications. The objective was to reduce hypothermia in babies <32 weeks of gestation in the delivery room to <40% using a checklist and sustain it for 6 months. Methods/UNASSIGNED:We created a delivery room checklist in 2012. Chart review established a baseline rate of hypothermia (<97°F). The team analyzed the checklist's effect on hypothermia from 2012 to 2018 and utilized numerous interventions to maintain compliance. Chi-square test and Fisher's exact test analyzed hypothermia and hyperthermia as a balancing measure. All calculations performed in SAS 9.3. Results/UNASSIGNED:The checklist reduced hypothermia from a baseline of 50% in 2011 (n = 104) to 33% in 2012 (n = 106). In 2013, the proportion of hypothermia slightly increased to 36% (n = 81). The year 2014 brought larger drift, and proportion of hypothermia increased to 44% (n = 117). In 2015, we reinforced the use of the checklist and proportion of hypothermia improved to 36% (n = 99). Further interventions through 2018 decreased hypothermia further to 14% to achieve statistical significance. Conclusions/UNASSIGNED:A checklist is a simple tool that may yield beneficial changes in practice and helped to decrease the proportion of neonatal hypothermia.

Cold stimulation reduces airway compromise in adults with dysphagia. However, there is no sufficient evidence to support its use in the pediatric population. The primary goal of this pilot study is to assess the effect of cold liquid on the pharyngeal swallow mechanism in preterm infants with dysphagia. We hypothesized that thermal stimulation from cold liquid will decrease the risk of airway compromise in dysphagic preterm infants. Nine preterm infants with clinical symptoms of dysphagia were included. Video fluoroscopic swallow studies were used to assess the swallowing mechanism of each participant. The occurrence of swallow dysfunctions under room temperature liquid swallows (RTS) vs. short period cold liquid swallows (CS) was compared. Paired t test was used to test significance. The occurrence of deep penetration (p = 0.007) and aspiration (p = 0.002) decreased significantly in the CS condition compared with the RTS condition. There was a trend of less nasopharyngeal reflux with CS but did not reach statistical significance (p = 0.084). No differences were noted for mild penetration (p = 0.824). CS reduced airway compromise in dysphagic preterm infants compared to RTS. These data provide important information regarding the immediate effects of CS on pharyngeal swallowing in preterm infants with dysphagia. However, further investigation regarding its sustained effects is required before introducing to clinical practice.

BACKGROUND: Intrauterine infection and inflammation during pregnancy, which leads to up-regulation of inflammatory cytokines and prostaglandin synthesis, has been implicated in the pathogenesis of preterm delivery and other pregnancy complications. Effective preventive and therapeutic strategies to reduce these outcomes are lacking to date. Pentoxifylline (PTX) is a non-specific phosphodiesterase inhibitor which raises intracellular cyclic adenosine monophosphate and decreases production of pro-inflammatory mediators while enhancing anti-inflammatory cytokines. We hypothesized that pentoxifylline will decrease lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production in human placental explants. METHODS: Placental explants derived from normal second trimester human placentas were treated with PTX, stimulated with LPS and cultured at 37 degrees C in 5% CO2. Conditioned media were assayed for pro- and anti-inflammatory mediators with multiplex immunoassays or ELISA, and explant tissues for mRNA with real time PCR. Means of PTX-treated and untreated samples were compared using paired t tests and Wilcoxon-signed rank tests. RESULTS: PTX preferentially inhibited placental expression and production of LPS-induced pro-inflammatory cytokines including TNF-alpha (25461 vs. 1908 pg/ml, p < 0.001), IL-1beta (2921 vs. 1067 pg/ml, p < 0.001) and IFN-gamma (2190 vs 427 pg/ml, p < 0.001) with relative preservation of anti-inflammatory mediators. The suppressive effects on LPS-induced placental inflammation were independent of the timing of PTX administration in relation to LPS-induced stimulation. CONCLUSION: Our study suggests that PTX attenuates the LPS-induced pro-inflammatory milieu in human placental explants. We speculate that PTX may have utility as a candidate anti-inflammatory agent for prophylaxis and/or treatment of human placental inflammation.

OBJECTIVE:Feeding neonates orally while on nasal continuous positive airway pressure (nCPAP) is a common practice. We hypothesize that pressurized airflow provided by nCPAP will alter the swallowing mechanism in neonates, increasing the risk of aspiration during oral feeding. STUDY DESIGN/METHODS:Infants receiving nCPAP with a RAM cannula and tolerating at least 50% of their feeding orally were included in the study (one term; six preterm infants). Each participant underwent a videofluoroscopic swallow study while on nCPAP and off nCPAP. A non-parametric signed-rank test was used for paired data. RESULT/RESULTS:The incidence of deep penetration (P=0.03) and aspiration (P=0.01) decreased significantly off-nCPAP compared with on-nCPAP. However, the incidence of mild penetration (P=0.65) and nasopharyngeal reflux (P=0.87) remained the same under both conditions. CONCLUSION/CONCLUSIONS:Oral feeding while on-nCPAP significantly increases the risk of laryngeal penetration and tracheal aspiration events. We recommend caution when initiating oral feedings on nCPAP.

OBJECTIVE: The prenatal detection rate of isolated fetal limb abnormalities ranges from 4 to 29.5%. Our aim was to determine the accuracy of a detailed ultrasound protocol in detecting isolated fetal limb abnormalities Methods: This is a retrospective study of infants born at our institution with isolated limb defects from 2009 to 2014. Antepartum and postpartum records were reviewed for genetic testing results. We routinely image both upper and lower extremities, including all long bones, hands, feet, fingers and toes. Posturing, muscular tone and movement are also noted. RESULTS: During the study period, there were 52 neonates born with isolated fetal limb abnormalities who had received a fetal anatomic survey in our ultrasound unit and 15 930 sonograms performed with normal findings; 36 out of the 52 had been prenatally diagnosed (detection rate 69%). The specificity of the protocol was 100% as there were no false positive cases, the positive predictive value was 100% and negative predictive value 99.8%. Forty-three of 52 neonates had normal genetic testing either prenatally or postnatally; 9 neonates did not undergo genetic testing. The average additional time required for this detailed protocol was <5 min for second trimester sonogram. CONCLUSION: A minimal investment in time for detailed evaluation of fetal limbs more than doubles the previously reported prenatal detection rate.

Problem: Intrauterine infections activate a proinflammatory cascade involving cytokines and other mediators that lead to preterm labor. Endotoxin tolerance (ET) is a phenomenon in which exposure to a dose of endotoxin renders tissues less responsive to subsequent exposures. The mechanism underlying ET is not fully understood. To our knowledge, no previous studies have elucidated the role of ET in human placenta. Using placental explants, we examined this phenomenon and whether exosomes play part in it. Method of Study: Placental explants from term and second-trimester pregnancies were cultured and exposed to low dose LPS for three days. Media were collected daily, and the explants were re-exposed to LPS. Cytochalasin-D (inhibitor of exosomes release and uptake) was added with LPS in some groups. TNF-aalpha and IL-10 in placental explants media were determined by ELISA. Exosomes were isolated from media by Total Exosome Isolation Kit, and miRNAs inside exosomes were analyzed by RT-PCR. Results: LPS treatment for 24 hours stimulated the secretion of placental pro-inflammatory cytokines. However, repeated treatment of the placental explants with LPS significantly reduced the subsequent pro-inflammatory effect, indicating ET. The anti-inflammatory cytokine, IL-10, was also induced by LPS; however, its levels were not affected on repeated LPS treatments. Cytochalasin-D treatment resulted in the loss of ET; nevertheless, it did not change IL-10 secretion. We observed that LPS increased exosomes secretion from placental explants. Moreover, miR-146a and others, which negatively modulate inflammatory response, were found higher in the LPS treated exosomes. Taking together, these findings suggest that ET is mediated by exosomes. Conclusion: This study illustrates, for the first time, that LPS induces ET in human placenta, and that exosomes mediate this phenomenon. We speculate that dysregulation of placental exosomes production, and thus tolerance to infection, might be linked to the exaggerated inflammatory response that leads to preterm labor

OBJECTIVE:Gentamicin is a common antibiotic used to treat sepsis in neonates. We hypothesize that obtaining routine gentamicin trough levels may not be necessary in low-risk, term infants. STUDY DESIGN:We performed a retrospective cohort study of term infants (n=346) treated with gentamicin in a single level III neonatal intensive care unit (NICU). The results of gentamicin trough levels and the correlation with risk factors and potential side effects were recorded. In addition, we conducted a survey of 75 academic NICUs across the United States regarding their gentamicin monitoring practice. RESULTS:Routine trough levels did not predict potential gentamicin toxicity in neonates with low risk factors. Regression analysis demonstrated a positive correlation between gentamicin trough levels and serum creatinine. The survey of the NICUs in the United States demonstrated significant inconsistency in gentamicin monitoring practice. CONCLUSION:Obtaining gentamicin trough levels guided by risk factors is more appropriate than obtaining routine trough levels in low-risk term neonates.

BACKGROUND: Midtrimester ultrasound is a valuable method for identifying asymptomatic women at risk for spontaneous preterm delivery (PTD). However, response to various treatments (cerclage, progestogen) has been variable in the clinical setting. It remains unclear how other biomarkers may be used to guide intervention strategies. OBJECTIVE: We applied an amniotic fluid inflammatory scoring system to determine if the degree of inflammation is associated with intervention efficacy in patients with midtrimester short cervix. STUDY DESIGN: Women carrying a singleton fetus between 16-24 weeks' gestation with a short cervix (/=8) or low (score <8) risk for inflammation. Gestational age at delivery was compared for each intervention and risk score status. Risk of delivering as a function of the remaining gestation was evaluated using modified Cox proportional hazards models with incorporation of methods to account for both left and right truncation bias. RESULTS: Ninety patients were included: 24 were in the nonintervention control group, 51 received cerclage, and 15 received 17OHP-C. Inflammation status at time of sampling influenced the efficacy of the treatment (P < .001). Compared to the nonintervention control group, in patients with low inflammation (score < 8), both cerclage (adjusted hazard ratio [HR], 2.86; 95% confidence interval [CI], 1.28-6.37) and 17OHP-C (HR, 3.11; 95% CI, 1.04-9.30) were associated with increased hazard of PTD. In contrast, in patients with high inflammation (score >/=8) both cerclage (HR, 0.22; 95% CI, 0.08-0.65) and 17OHP-C (HR, 0.20; 95% CI, 0.05-0.81) were associated with lower hazard of delivering preterm. CONCLUSION: Cerclage placement or administration of 17OHP-C therapy for midtrimester short cervix for PTD prevention appears beneficial only in the subset of patients with high inflammation. Knowledge of the amniotic fluid inflammatory status may aid in guiding the appropriate therapy for women presenting with midtrimester short cervix who are at increased risk of PTD.

Preterm birth (PTB), defined as any delivery occurring prior to the completion of 37 weeks' gestation, currently accounts for 11-12% of all births in the United States. Maternal genito-urinary infections account for up to 40% of all PTBS and induce a pro-inflammatory state in the host. The potent vasoconstrictor Endothelin-1 (ET-1) is known to be upregulated in the setting of infection, and elicits its effect by binding to the ETA receptor. We have previously shown that antagonism of the ETA receptor with BQ-123 is capable of preventing LPS-induced PTB in mice. We hypothesize that the administration of BQ-123 post LPS exposure will dismantle a positive feedback loop observed with pro-inflammatory cytokines upstream of ET-1. On GD 15.5, pregnant C57BL/6 mice were injected with PBS, LPS, BQ-123, or LPS+BQ-123. Changes at both the level of transcription and translation were observed in uterus and placenta in the ET-1 axis and in pro- and anti-inflammatory cytokines over the course of 12h. We discovered that BQ-123, when administered 10h post LPS, is capable of increasing production of uterine and placental Interleukin-10, causing a shift away from the pro-inflammatory state. We also observed that antagonism of the ETA receptor decreased IL-1β and TNFα in the placenta while also decreasing transcription of ET-1 in the uterus. Our results reinforce the role of ET-1 at the maternal fetal interface and highlight the potential benefit of ETA receptor blockade via the suppression of ET-1, and induction of a Th2 cytokine dominant state.