Faculty Bibliography

In the maternal circulation, apoB-containing low-density lipoproteins (LDL) and apoA1-containing high-density lipoproteins (HDL) transport lipids. The production of lipoproteins in the placenta has been suggested, but the directionality of release has not been resolved. We compared apolipoprotein concentrations and size-exclusion chromatography elution profiles of lipoproteins in maternal/fetal circulations, and in umbilical arteries/veins; identified placental lipoprotein-producing cells; and studied temporal induction of lipoprotein-synthesizing machinery during pregnancy. We observed that maternal and fetal lipoproteins are different with respect to concentrations and elution profiles. Surprisingly, concentrations and elution profiles of lipoproteins in umbilical arteries and veins were similar indicating their homeostatic control. Human placental cultures synthesized apoB100-containing LDL-sized and apoA1-containing HDL-sized particles. Immunolocalization techniques revealed that ApoA1 was present mainly in syncytiotrophoblasts. MTP, a critical protein for lipoprotein assembly, was in these trophoblasts. ApoB was in the placental stroma indicating that trophoblasts secrete apoB-containing lipoproteins into the stroma. ApoB and MTP expressions increased in placentas from the 2nd trimester to term, whereas apoA1 expression was unchanged. Thus, our studies provide new information regarding the timing of lipoprotein gene induction during gestation, the cells involved in lipoprotein assembly and the gel filtration profiles of human placental lipoproteins. Next, we observed that mouse placenta produces MTP, apoB100, apoB48 and apoA1. The expression of genes gradually increased and peaked in late gestation. This information may be useful in identifying transcription factors regulating the induction of these genes in gestation and the importance of placental lipoprotein assembly in fetal development.

OBJECTIVE: Human milk (HM) has antibacterial properties due to the presence of immune-modulators, including lactoferrin (LF). This study will determine effect(s) of HM maturation, fortification, and storage conditions on LF levels and its antibacterial properties. STUDY DESIGN/METHODS:. RESULTS: The highest level of LF in preterm HM was observed in the first week of lactation. However, storage of preterm HM at 4°C decreased LF levels significantly. Both LF levels and antibacterial activity in preterm HM was lower compared with term HM, but significantly higher than donor HM even after HM-based fortification. LF supplementation of donor HM improved its antibacterial activity. CONCLUSION/CONCLUSIONS: Preterm infants fed donor HM, formula, or stored HM at 4°C may benefits from LF supplementation to improve HM antibacterial properties. KEY POINTS/CONCLUSIONS:· Milk LF levels vary with storage and maturity.. · Donor milk is deficient in LF even after adding HM-based fortification.. · Donor HM and formula fed infants may benefit from LF..

Neonates born prematurely (<37 weeks of gestation) are at a significantly increased risk of developing inflammatory conditions associated with high mortality rates, including necrotizing enterocolitis, bronchopulmonary dysplasia, and hypoxic-ischemic brain damage. Recently, research has focused on characterizing the content of extracellular vesicles (EVs), particularly microRNAs (miRNAs), for diagnostic use. Here, we describe the most recent work on EVs-miRNAs biomarkers discovery for conditions that commonly affect premature neonates.

BACKGROUND/UNASSIGNED:Treatment for head and neck cancer (HNC) such as radiotherapy (RT) can lead to numerous acute and chronic head and neck sequelae, including dental caries. The goal of the present study was to measure 2-y changes in dental caries after radiotherapy in patients with HNC and test risk factors for caries increment. METHODS/UNASSIGNED:Cancer and dental disease characteristics, demographics, and oral health practices were documented before and 6, 12, 18, and 24 mo after the start of RT for 572 adult patients with HNC. Patients were eligible if they were age 18 y or older, diagnosed with HNC, and planned to receive RT for treatment of HNC. Caries prevalence was measured as decayed, missing, and filled surfaces (DMFS). The association between change in DMFS and risk factors was evaluated using linear mixed models. RESULTS/UNASSIGNED:= 164), lower salivary flow at follow-up visits was associated with increased DMFS. CONCLUSION/UNASSIGNED:Increased caries is a complication soon after RT in HNC. Fluoride, oral hygiene, dental insurance, and education level had the strongest association with caries increment after radiotherapy to the head and neck region. Thus, intensive oral hygiene measures, including fluoride and greater accessibility of dental care, may contribute to reducing the caries burden after RT in HNC. KNOWLEDGE TRANSFER STATEMENT/UNASSIGNED:The results of this study can be used by clinicians when deciding how to minimize oral complications related to cancer therapy for patients with head and neck cancer. Identification of modifiable factors (e.g., oral hygiene and prescription fluoride compliance) associated with increased caries risk can minimize radiation caries burden.

BACKGROUND:Pregnancy represents a unique challenge for the maternal-fetal immune interface, requiring a balance between immunosuppression, which is essential for the maintenance of a semi-allogeneic fetus, and pro-inflammatory host defense to protect the maternal-fetal interface from invading organisms. Adaptation to repeated inflammatory stimuli (endotoxin tolerance) may be critical in preventing inflammation-induced preterm birth resulting from exaggerated maternal inflammatory responses to mild/moderate infections that are common during pregnancy. However, the exact mechanisms contributing to the maintenance of tolerance to repeated infections are not completely understood. miRNAs play important roles in pregnancy, with several miRNAs implicated in gestational tissue function, as well as in pathologic pregnancy conditions. miRNA-519c, a member of the C19MC cluster, is a human-specific miRNA mainly expressed in the placenta. However, its role in pregnancy is largely unknown. OBJECTIVES/OBJECTIVE:To explore the role of "endotoxin tolerance" failure in the pathogenesis of an exaggerated inflammatory response often seen in inflammation-mediated preterm birth. In this study, we investigated the role of miRNA-519c, a placenta-specific miRNA, as a key regulator of endotoxin tolerance at the maternal-fetal interface. STUDY DESIGN/METHODS:-trimester placentas were treated with LPS. After 24 hours, the conditioned media was collected for analysis, and the placental explants were re-exposed to repeated doses of LPS for 3 days. The supernatant was analyzed for inflammatory markers, presence of extracellular vesicles (EVs) and microRNAs. To study the possible mechanism of action of the microRNAs, we evaluated the phosphodiesterase 3 B (PDE3B) pathway involved in TNF-α production using a miRNAs mimic and PDE3B siRNA transfection. Finally, we analyzed human placental samples from different gestational ages and from women affected by inflammation-associated pregnancies. RESULTS:Our data showed that repeated exposure of the human placenta to endotoxin challenges induced a tolerant phenotype characterized by decreased TNF-α and upregulated IL-10 levels. This reaction was mediated by the placenta-specific miRNA-519c packaged within placental EVs. LPS treatment increased the EVs that were positive for the exosome tetraspanin markers, namely CD9, CD63, and CD81, and secreted primarily by trophoblasts. Primary human trophoblast cells transfected with miR-519c mimic decreased PDE3B. While lack of PDE3B, achieved by siRNA transfection, resulted in a decreased TNF-α production. These data supported the hypothesis that the anti-inflammatory action of miRNA-519c was mediated by a downregulation of the phosphodiesterase 3 B pathway, leading to inhibition of TNF-α production. Furthermore, human placentas from normal and inflammation-associated pregnancies demonstrated that decreased placental miRNA-519c level was linked to infection-induced inflammatory pathologies during pregnancy. CONCLUSION/CONCLUSIONS:We identified miRNA-519c, a human placenta-specific miRNA, as a novel regulator of immune adaptation associated with infection-induced preterm birth at the maternal-fetal interface. Our study can serve as a basis for future experiments to explore the potential use of miRNA-519c as a biomarker for infection-induced preterm birth.

OBJECTIVE: There are limited published data on the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus from mothers to newborns through breastfeeding or from breast milk. The World Health Organization released guidelines encouraging mothers with suspected or confirmed COVID-19 to breastfeed as the benefits of breastfeeding outweighs the possible risk of transmission. The objective of this study was to determine if SARS-CoV-2 was present in the breast milk of lactating mothers who had a positive SARS-CoV-2 nasopharyngeal swab test prior to delivery, and the clinical outcomes for their newborns. STUDY DESIGN/METHODS:by two-step reverse transcription polymerase chain reaction. Additionally, the clinical characteristics of the maternal newborn dyad, results of nasopharyngeal SARS-CoV-2 testing, and neonatal follow-up data were collected. RESULTS: A total of 19 mothers were included in the study and their infants who were all fed breast milk. Breast milk samples from 18 mothers tested negative for SARS-CoV-2, and 1 was positive for SARS-CoV-2 RNA. The infant who ingested the breast milk that tested positive had a negative nasopharyngeal test for SARS-CoV-2, and had a benign clinical course. There was no evidence of significant clinical infection during the hospital stay or from outpatient neonatal follow-up data for all the infants included in this study. CONCLUSION/CONCLUSIONS: In a small cohort of SARS-CoV-2 positive lactating mothers giving birth at our institution, most of their breast milk samples (95%) contained no detectable virus, and there was no evidence of COVID-19 infection in their breast milk-fed neonates. KEY POINTS/CONCLUSIONS:· Breast milk may rarely contain detectable SARS-CoV-2 RNA and was not detected in asymptomatic mothers.. · Breast milk with detectable SARS-CoV-2 RNA from a symptomatic mother had no clinical significance for her infant.. · Breast feeding with appropriate infection control instructions appears to be safe in mother with COVID infection..

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.

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.