Hepatoviruses promote very-long-chain fatty acid and sphingolipid synthesis for viral RNA replication and quasi-enveloped virus release.

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl-coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides. Our data provide insight into how a pathogenic virus alters lipid flux in infected hepatocytes and demonstrate a distinction between lipid species required for viral RNA synthesis versus nonlytic quasi-enveloped virus release.

A truncated progesterone receptor (PR-M) localizes to the mitochondrion and controls cellular respiration.

The cDNA for a novel truncated progesterone receptor (PR-M) was previously cloned from human adipose and aortic cDNA libraries. The predicted protein sequence contains 16 unique N-terminal amino acids, encoded by a sequence in the distal third intron of the progesterone receptor PR gene, followed by the same amino acid sequence encoded by exons 4 through 8 of the nuclear PR. Thus, PR-M lacks the N terminus A/B domains and the C domain for DNA binding, whereas containing the hinge and hormone-binding domains. In this report, we have localized PR-M to mitochondria using immunofluorescent localization of a PR-M-green fluorescent protein (GFP) fusion protein and in Western blot analyses of purified human heart mitochondrial protein. Removal of the putative N-terminal mitochondrial localization signal obviated association of PR-M with mitochondria, whereas addition of the mitochondrial localization signal to green fluorescent protein resulted in mitochondrial localization. Immunoelectron microscopy and Western blot analysis after mitochondrial fractionation identified PR-M in the outer mitochondrial membrane. Antibody specificity was shown by mass spectrometry identification of a PR peptide in a mitochondrial membrane protein isolation. Cell models of overexpression and gene silencing of PR-M demonstrated a progestin-induced increase in mitochondrial membrane potential and an increase in oxygen consumption consistent with an increase in cellular respiration. This is the first example of a truncated steroid receptor, lacking a DNA-binding domain that localizes to the mitochondrion and initiates direct non-nuclear progesterone action. We hypothesize that progesterone may directly affect cellular energy production to meet the increased metabolic demands of pregnancy.

Apoptosis in the chorion of fetal membranes in preterm premature rupture of membranes.

We sought to determine if apoptosis in the chorion of fetal membranes was increased in patients with preterm premature rupture of membranes (PPROM) with histological chorioamnionitis. Using the TUNEL (terminal uridine deoxynucleotidyl transferase dUTP nick-end labeling) method (ApopTag Plus kit; Oncor, Gaithersburg, MD), apoptosis was quantified. Of the 47 subjects with PPROM, 18 lacked sufficient chorion for quantification (confirmed by cytokeratin staining). In the remaining 30 subjects, fetal membranes with and without chorioamnionitis were examined and apoptosis was quantified. There were no differences in maternal age, race, insurance, cesarean rate, or gestational age between groups. The chorion of fetal membranes from PPROM patients with chorioamnionitis had significantly more apoptotic nuclei than those without chorioamnionitis (19.1 versus 0.8; P= .005). Of the 17 subjects excluded for absence of chorion, 16 (94%) had at least moderate chorioamnionitis. This investigation suggests that apoptosis is accelerated in the chorion of PPROM subjects with chorioamnionitis. Absence of the chorion in 37% of subjects is supportive of the hypothesis that inflammation accelerates cell death and destruction of the chorion.

Progesterone protects fetal chorion and maternal decidua cells from calcium-induced death.

OBJECTIVE: The purpose of this study was to determine whether progesterone exerts a protective effect in chorion and decidua cells when exposed to calcimycin. STUDY DESIGN: Fetal membrane samples were collected from term elective repeat cesarean deliveries and chorion and decidua cells that are separated and cultured. Cells were pretreated with progesterone and exposed to calcimycin. Cell viability was determined, and percent cell viability was calculated. RESULTS: Exposure to calcimycin resulted in a reduction of cell viability in both chorion and decidua cells in a dose-dependent fashion. In chorion and decidua cells, progesterone pretreatment followed by calcimycin increased cell viability compared with calcimycin treatment alone (chorion, 67%, vs controls, 24%; P < .001; decidua, 58%, vs controls, 35%; P < .001). The progesterone receptor antagonist, RTI 6413-49a, blocked the protective effect of progesterone in both chorion and decidua cells. CONCLUSION: These preliminary results suggest that progesterone may provide a protective effect in fetal membrane cells and that this effect may be mediated through the progesterone receptor.

Thrombospondin-1 and thrombospondin-2 mRNA and TSP-1 and TSP-2 protein expression in uterine fibroids and correlation to the genes COL1A1 and COL3A1 and to the collagen cross-link hydroxyproline.

Uterine fibroids are composed of altered collagen fibrils and represent an arrested response to injury-initiating fibrosis. In many tissues, TSP-1 is secreted by adult macrophages and monocytes upon wounding and is involved in the activation of transforming growth factor beta. In the absence of TSP-1, the orchestrated process of wound healing is impaired. The authors obtained tissue from the edge and center of fibroids at the time of hysterectomy and compared them with adjacent myometrium. The pattern of TSP-1 and TSP-2 expression was correlated to that of COL1A1 and COL3A1. Collagen and hydroxyproline were increased in fibroids. Thrombospondin-1 was consistently underexpressed in both the edge and center of the fibroids, while COL1A1 and COL3A1 were consistently overexpressed. However, TSP-2 was inconsistently expressed. These findings lead to the conclusion that the underexpression of TSP-1 may contribute to the overall development of uterine fibroids.

Association of maternal IL-1 receptor antagonist intron 2 gene polymorphism and preterm birth.

OBJECTIVE: This study was undertaken to determine whether the interleukin-1 receptor antagonist (IL-1RN) variable number tandem repeat polymorphism is associated with preterm birth. STUDY DESIGN: A case-control study was performed. Cases (n = 95) delivered before 37 weeks after preterm labor (PTL) or preterm premature rupture of membranes (PPROM) and controls (n = 105) delivered after 37 weeks. Maternal DNA was genotyped by polymerase chain reaction for a length polymorphism in intron 2 of the IL-1RN gene. RESULTS: There was no significant difference in maternal age, ethnicity, insurance status, or parity between groups. Allele and genotype frequencies did not differ significantly from that expected under Hardy-Weinberg equilibrium (P = .59) in the total group as well as study groups. Of the 95 cases, 26.8% had at least 1 copy of allele 2 present compared with 12.4% in the control group (P < .0004). CONCLUSION: Maternal carriage of at least 1 copy of the IL-1RN allele 2 appears to be associated with increased risk of preterm birth.

Circulating angiogenic factors and abnormal uterine artery Doppler velocimetry in the second trimester.

OBJECTIVE: Circulating angiogenic growth factors (such as vascular endothelial growth factor [VEGF] and placental growth factor [PlGF]) and their interaction may be associated with vascular remodeling of spiral arteries in normal pregnancy. Soluble Flt-1, an antagonist of both VEGF and PlGF, has been shown to be increased, while PlGF is decreased in women prior to the onset of preeclampsia. The purpose of this study was to compare maternal soluble Flt-1 and PlGF levels in the second trimester with a marker of abnormal placentation, abnormal uterine artery Doppler (UAD). METHOD: A prospective cohort of women, 16 to 24 weeks estimated gestational age (EGA), with singleton pregnancies, underwent UAD and phlebotomy. Maternal soluble Flt-1 and free PlGF were measured by ELISA in samples from women with abnormal UAD with a group, controlled for EGA, with normal UAD. Mann-Whitney Rank-Sum test was used to compare maternal serum levels of both soluble Flt-1 and PlGF between women with abnormal uterine artery Doppler versus women with normal uterine artery Doppler. RESULTS: Of the 222 study subjects enrolled, 34 (15%) had abnormal UAD. The mean EGA at enrollment of subjects in each group was 18 weeks. There was no difference in PlGF between subjects with abnormal UAD (median, 191 pg/mL; range, 187 to 337 pg/mL) versus controls (median, 171 pg/mL; range, 169 to 289 pg/mL) (p = 0.59) or soluble Flt-1 (median, 780 pg/mL; range, 280 to 3200 pg/mL) or between subjects with abnormal UAD versus controls (median, 720 pg/mL; range, 220 to 1980 pg/mL) (p = 0.36). CONCLUSION: Concentrations of maternal soluble Flt-1 and free PlGF in the second trimester do not appear to be altered in women with abnormal UAD. This suggests that these biochemical markers are independent of the increased placental resistance seen with abnormal uterine artery Doppler.

Characterization of progesterone receptor isoform expression in fetal membranes.

OBJECTIVE: To quantify expression of progesterone receptor (PR) messenger RNA (mRNA) isoforms in fetal membranes, and to determine whether these levels change in culture. STUDY DESIGN: Placentas from women undergoing term cesarean delivery before labor were collected. Layers of amnion, chorion, and decidua were separated manually, enzymatically digested, and separated further with the use of a density gradient. RNA was extracted immediately and after culture for 48 hours, then analyzed by quantitative reverse transcription polymerase chain reaction for PR-A, PR-B, and beta-2 microglobulin mRNA expression. Separation of cell types was confirmed by immunohistochemistry. RESULTS: PR isoform expression was identified in fetal membranes, with levels highest in decidua and below the limits of detection in amnion. The ratio of PR-A/PR-B mRNA was not significantly different between cell layers. PR mRNA isoform levels did not differ significantly in fresh versus cultured cells. CONCLUSION: Quantitative reverse transcription polymerase chain reaction was used to quantitate expression of PR mRNA isoforms in cells of fetal membranes and to validate systems for further study of PR with respect to inflammation, infection, and preterm delivery.