Hepcidin, an Iron Regulatory Hormone of Innate Immunity, is Differentially Expressed in Premature Fetuses with Early-Onset Neonatal Sepsis.

OBJECTIVE: Hepcidin, a mediator of innate immunity, binds the iron exporter ferroportin, leading to functional hypoferremia through intracellular iron sequestration. We explored hepcidin-ferroportin interactions in neonates clinically diagnosed with early-onset neonatal sepsis (EONS). STUDY DESIGN: Hepcidin and interleukin (IL)-6 were quantified by enzyme-linked immunosorbent assay (ELISA) in 92 paired cord blood-maternal blood samples in the following groups: "Yes" EONS (n = 41, gestational age [GA] 29 ± 1 weeks) and "No" EONS (n = 51, GA 26 ± 1 weeks). Placental hepcidin and ferroportin expression were evaluated by immunohistochemistry and real-time-polymerase chain reaction (RT-PCR). Liver hepcidin and ferroportin expression patterns were ascertained in autopsy specimens of neonates (n = 8) who died secondary to culture-proven sepsis. RESULTS: Cord blood hepcidin was significantly elevated (GA corrected, p = 0.018) and was positively correlated with IL-6 (r = 0.379, p = 0.001) in EONS. Hepcidin localized at syncytiotrophoblast and fetal vascular endothelium. Placental ferroportin, but not hepcidin mRNA correlated with cord blood hepcidin levels (r = 0.46, p = 0.039) and funisitis severity (r = 0.50, p = 0.018). Newborns who died from sepsis (n = 4) had higher hepatic hepcidin and iron sequestration, but lower ferroportin staining than those who died of nonsepsis causes (n = 4). CONCLUSION: Premature fetuses with EONS have elevated circulating hepcidin, likely related to lower placenta and liver ferroportin expression. Fetal hepcidin-ferroportin interaction appears to play a role in EONS pathophysiology independent of maternal response to intrauterine inflammation.

Dissecting the mechanisms of Notch induced hyperplasia.

The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch-CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non-autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed-forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross-regulatory relationships are important in coordinating the pattern of hyperplasia.

Serum and Urine Thioflavin-T-Enhanced Fluorescence in Severe Preeclampsia.

Common features of amyloid-like proteotoxic aggregates are the ability to bind Congo red (congophilia) and to induce fluorescence of thioflavin-T (ThT). Based on the prior discovery that women with preeclampsia exhibit urine congophilia, we proposed that amyloid-like protein aggregates present in urine also circulate in the bloodstream and this feature is linked to disease severity and clinical phenotype. ThT fluorescence was investigated in 217 paired serum and urine samples from women with severe features of preeclampsia (n=101; median [interquartile range] gestational age [GA], 32 [29-35] weeks), mild features of preeclampsia (n=22; GA, 36 [36-37] weeks), chronic hypertension (n=15; GA, 38 [37-39] weeks), healthy pregnant controls (n=57; GA, 39 [38-39] weeks), and nonpregnant controls (n=22). Serum and urine fluorescence attributable to advanced glycation end products was measured in the same samples with correction for autofluorescence. There were no GA-related changes in ThT fluorescence, although near-term serum ThT fluorescence increased compared with nonpregnant state. Compared with healthy pregnant controls, serum and urine ThT fluorescence was increased in severe features of preeclampsia (P<0.001 for both) but not in mild features of preeclampsia or chronic hypertension. Except for chronic hypertension, advanced glycation end products-related fluorescence of serum or urine did not differ from controls. Urine congophilia had a stronger relationship with preeclampsia severity compared with either urine or serum ThT fluorescence. However, serum ThT fluorescence was independently associated with clinical features of hemolysis, elevated liver enzyme levels, and low platelet levels syndrome (P=0.003). We demonstrate that ThT fluorescence, a marker of amyloid-like aggregates, is increased in serum of women with preeclampsia and likely because of their cytotoxicity associated with hemolysis, elevated liver enzyme levels, and low platelet levels syndrome.

Evidence for participation of neutrophil gelatinase-associated lipocalin/matrix metalloproteinase-9 (NGAL•MMP-9) complex in the inflammatory response to infection in pregnancies complicated by preterm birth.

PROBLEM: Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in neutrophils and involved in innate immunity by sequestering iron. NGAL's ability to complex with matrix metalloproteinase-9 (MMP-9) and extend its gelatinolytic activity led us to investigate its role in pregnancies complicated by preterm birth (PTB) and intra-amniotic infection/inflammation (IAI). METHOD OF STUDY: We assayed the amniotic fluid (AF) levels of NGAL and MMP-9 in 308 women that had a clinically indicated amniocentesis and a normal pregnancy outcome or PTB. qRT-PCR was employed to determine NGAL mRNA expression of placental villous trophoblast and amniochorion. Immunohistochemistry was used for cellular localization. RESULTS: AF NGAL levels were gestational age-regulated. Women with IAI and PTB had significantly higher levels of NGAL, MMP-9 and NGAL•MMP-9 complex. CONCLUSION: The amniochorion is a source of NGAL and similarly to other inflammatory conditions, this protein may augment the collagenolytic effect of MMP-9 and modulate host-microbe interactions in pregnancies complicated by IAI.

Evidence-based guidelines in labor management.

Evidence-based care of women in labor requires a thorough understanding of both "normal" and abnormal labor progress. In response to the growing cesarean delivery rate for dystocia at our institution, a multidisciplinary team of attending physicians, nurse-midwives, resident physicians, and nurses was established to review the literature and create evidence-based guidelines. This article describes the background literature and consensus guidelines reached for the diagnosis of active phase labor, active phase arrest, second-stage arrest, protraction of the active phase, and failed induction of labor. Our review illustrates that slower labor patterns than traditionally described often result in a vaginal delivery without unacceptable increases in maternal or neonatal morbidity.

Multiple Tyrosine Residues Contribute to GABA Binding in the GABA(C) Receptor Binding Pocket.

The ligand binding site of Cys-loop receptors is dominated by aromatic amino acids. In GABA(C) receptors, these are predominantly tyrosine residues, with a number of other aromatic residues located in or close to the binding pocket. Here we examine the roles of these residues using substitution with both natural and unnatural amino acids followed by functional characterization. Tyr198 (loop B) has previously been shown to form a cation-π interaction with GABA; the current data indicate that none of the other aromatic residues form such an interaction, although the data indicate that both Tyr102 and Phe138 may contribute to stabilization of the positively charged amine of GABA. Tyr247 (loop C) was very sensitive to substitution and, combined with data from a model of the receptor, suggest a π-π interaction with Tyr241 (loop C); here again functional data show aromaticity is important. In addition the hydroxyl group of Tyr241 is important, supporting the presence of a hydrogen bond with Arg104 suggested by the model. At position Tyr102 (loop D) size and aromaticity are important; this residue may play a role in receptor gating and/or ligand binding. The data also suggest that Tyr167, Tyr200, and Tyr208 have a structural role while Tyr106, Trp246, and Tyr251 are not critical. Comparison of the agonist binding site "aromatic box" across the superfamily of Cys-loop receptors reveals some interesting parallels and divergences.

Transducing agonist binding to channel gating involves different interactions in 5-HT3 and GABAC receptors.

5-hydroxytryptamine (5-HT)3 and gamma-aminobutyric acid, type C (GABAC) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which also includes nicotinic acetylcholine, GABAA, and glycine receptors. The details of how agonist binding to these receptors results in channel opening is not fully understood but is known to involve charged residues at the extracellular/transmembrane interface. Here we have examined the roles of such residues in 5-HT3 and GABAC receptors. Charge reversal experiments combined with data from activation by the partial agonist beta-alanine show that in GABAC receptors there is a salt bridge between Glu-92 (in loop 2) and Arg-258 (in the pre-M1 region), which is involved in receptor gating. The equivalent residues in the 5-HT3 receptor are important for receptor expression, but charge reversal experiments do not restore function, indicating that there is not a salt bridge here. There is, however, an interaction between Glu-215 (loop 9) and Arg-246 (pre-M1) in the 5-HT3 receptor, although the coupling energy determined from mutant cycle analysis is lower than might be expected for a salt bridge. Overall the data show that charged residues at the extracellular/transmembrane domain interfaces in 5-HT3 and GABAC receptors are important and that specific, but not equivalent, molecular interactions between them are involved in the gating process. Thus, we propose that the molecular details of interactions in the transduction pathway between the binding site and the pore can differ between different Cys-loop receptors.