Longitudinal analysis of maternal plasma apolipoproteins in pregnancy: a targeted proteomics approach.

Minimally invasive diagnostic tests are needed in obstetrics to identify women at risk for complications during delivery. The apolipoproteins fluctuate in complexity and abundance in maternal plasma during pregnancy and could be incorporated into a blood test to evaluate this risk. The objective of this study was to examine the relative plasma concentrations of apolipoproteins and their biochemically modified subtypes (i.e. proteolytically processed, sialylated, cysteinylated, dimerized) over gestational time using a targeted mass spectrometry approach. Relative abundance of modified and unmodified apolipoproteins A-I, A-II, C-I, C-II, and C-III was determined by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in plasma prospectively collected from 11 gravidas with uncomplicated pregnancies at 4-5 gestational time points per patient. Apolipoproteins were readily identifiable by spectral pattern. Apo C-III(2) and Apo C-III(1) (doubly and singly sialylated Apo C-III subtypes) increased with gestational age (r(2)>0.8). Unmodified Apo A-II, Apo C-I, and Apo C-III(0) showed no correlation (r(2) = 0.01-0.1). Pro-Apo C-II did not increase significantly until third trimester (140 ± 13% of first trimester), but proteolytically cleaved, mature Apo C-II increased in late pregnancy (702 ± 130% of first trimester). Mature Apo C-II represented 6.7 ± 0.9% of total Apo C-II in early gestation and increased to 33 ± 4.5% in third trimester. A label-free, semiquantitative targeted proteomics approach was developed using LTQ-Orbitrap mass spectrometry to confirm the relative quantitative differences observed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in Apo C-III and Apo C-II isoforms between first and third trimesters. Targeted apolipoprotein screening was applied to a cohort of term and preterm patients. Modified Apo A-II isoforms were significantly elevated in plasma from mothers who delivered prematurely relative to term controls (p = 0.02). These results support a role for targeted proteomics profiling approaches in monitoring healthy pregnancies and assessing risk of adverse obstetric outcomes.

The effect of magnesium sulfate on the activity of matrix metalloproteinase-9 in fetal cord plasma and human umbilical vein endothelial cells.

OBJECTIVE: Clinical evidence suggests that magnesium sulfate may reduce the risk of fetal neurologic injury in preterm delivery. Matrix metalloproteinase-9 (MMP-9) levels are elevated in preterm labor patients. There is evidence that MMP-9 may break down the blood-brain barrier in humans, causing cytokine mediated cell injury. Our objective was to determine whether the addition of magnesium sulfate attenuates activity of MMP-9, a complex zinc-dependent enzyme, in fetal cord plasma. STUDY DESIGN: We collected cord plasma in 6 term, unlabored patients. Using enzyme-linked immunosorbent assay, we measured the activity of MMP-9 with varying concentrations of magnesium sulfate added in vitro. Results were verified using a human umbilical cord vein endothelial cell (HUVEC) line. RESULTS: Addition of physiologic doses of magnesium sulfate (0.07 mg/mL) resulted in a 25% decrease in active MMP-9 (P = .03). In a HUVEC line, magnesium sulfate resulted in a 32% decrease in MMP-9 activity (P = .00012). CONCLUSION: The addition of magnesium sulfate attenuated MMP-9 activity in cord plasma and in a HUVEC line.

Group B streptococcus serotype prevalence in reproductive-age women at a tertiary care military medical center relative to global serotype distribution.

BACKGROUND: Group B Streptococcus (GBS) serotype (Ia, Ib, II-IX) correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan), the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations. METHODS: To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps) genes associated with pathogen virulence. RESULTS: Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively). A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p < 0.005). The number of non-typeable isolates was significantly lower in the study (p < 0.005). CONCLUSION: This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades.

Vitamin D deficiency in early pregnancy.

OBJECTIVE: Vitamin D deficiency is a common problem in reproductive-aged women in the United States. The effect of vitamin D deficiency in pregnancy is unknown, but has been associated with adverse pregnancy outcomes. The objective of this study was to analyze the relationship between vitamin D deficiency in the first trimester and subsequent clinical outcomes. STUDY DESIGN: This is a retrospective cohort study. Plasma was collected in the first trimester from 310 nulliparous women with singleton gestations without significant medical problems. Competitive enzymatic vitamin D assays were performed on banked plasma specimens and pregnancy outcomes were collected after delivery. Logistic regression was performed on patients stratified by plasma vitamin D concentration and the following combined clinical outcomes: preeclampsia, preterm delivery, intrauterine growth restriction, gestational diabetes, and spontaneous abortion. RESULTS: Vitamin D concentrations were obtained from 235 patients (mean age 24.3 years, range 18-40 years). Seventy percent of our study population was vitamin D insufficient with a serum concentration less than 30 ng/mL (mean serum concentration 27.6 ng/mL, range 13-71.6 ng/mL). Logistic regression was performed adjusting for age, race, body mass index, tobacco use, and time of year. Adverse pregnancy outcomes included preeclampsia, growth restriction, preterm delivery, gestational diabetes, and spontaneous abortion. There was no association between vitamin D deficiency and composite adverse pregnancy outcomes with an adjusted odds ratio of 1.01 (p value 0.738, 95% confidence intervals 0.961-1.057). CONCLUSION: Vitamin D deficiency did not associate with adverse pregnancy outcomes in this study population. However, the high percentage of affected individuals highlights the prevalence of vitamin D deficiency in young, reproductive-aged women.

Expression patterns of progesterone receptor membrane components 1 and 2 in endometria from women with and without endometriosis.

Endometriosis is a hormone-dependent inflammatory condition associated with pain and infertility. A growing body of evidence supports attenuated secretory-phase progesterone responsiveness in women with this disease. Herein, we compare the expression of progesterone receptor membrane components (PGRMC) 1 and 2 in eutopic endometrium from 11 women with laparoscopically and/or histologically proven stage III/IV endometriosis and 23 disease-free women. Menstrual cycle phase was determined using a combination of reported cycle day, serum hormone profile, and endometrial histologic dating. The PGRMC-1 (fold change -3.3; P < .05) and PGRMC-2 (fold-change -8.8; P < .05) gene expression were significantly downregulated in secretory phase, eutopic endometrium from women with endometriosis. Immunohistochemistry demonstrated decreased PGRMC-1 and PGRMC-2 protein expression in the secretory phase endometrial stroma cells of women with endometriosis. Consistent with the preclinical work of others, our results reflect downregulation of endometrial PGRMC-1 and PGRMC-2 expression in secretory phase endometrium from women with advanced stage endometriosis. Understanding the molecular mechanisms of attenuated progesterone action in endometriosis has important diagnostic and therapeutic implications.

Effect of dexamethasone administered with magnesium sulfate on inflammation-mediated degradation of the blood-brain barrier using an in vitro model.

Patients at risk for preterm delivery are frequently administered both antenatal steroids for fetal maturation and magnesium sulfate for neuroprotection. In this study, we investigate whether steroids coadministered with magnesium sulfate preserve blood-brain barrier integrity in neuroinflammation. Human umbilical vein endothelial cells were grown in astroglial conditioned media in a 2-chamber cell culture apparatus. Treatment with tumor necrosis factor-α (TNF-α) or catalytically active recombinant matrix metalloproteinase 9 (MMP-9) simulated neuroinflammation. Membrane integrity was assessed by zona occludens 1 (ZO-1) immunoreactivity, permeability to fluorescently conjugated dextran, and transendothelial electrical resistance (TEER). The TNF-α and MMP-9 treatment increased the rate of dextran transit, decreased TEER, and decreased ZO-1 immunoreactivity at junctional interfaces. Dexamethasone pretreatment alone or in combination with 0.5 mmol/L magnesium sulfate preserved monolayer integrity after inflammatory insult. Magnesium sulfate alone was not protective. This study supports a possible interaction between steroids and magnesium in neuroprotection.

Aberrant glycosylation of plasma proteins in severe preeclampsia promotes monocyte adhesion.

Glycosylation of plasma proteins increases during pregnancy. Our objectives were to investigate an anti-inflammatory role of these proteins in normal pregnancies and determine whether aberrant protein glycosylation promotes monocyte adhesion in preeclampsia. Plasma was prospectively collected from nonpregnant controls and nulliparous patients in all 3 trimesters. Patients were divided into cohorts based on the applicable postpartum diagnosis. U937 monocytes were preconditioned with enzymatically deglycosylated plasma, and monocyte adhesion to endothelial cell monolayers was quantified by spectrophotometry. Plasma from nonpregnant controls, first trimester normotensives, and first trimester patients with mild preeclampsia inhibited monocyte-endothelial cell adhesion (P < .05), but plasma from first trimester patients with severe preeclampsia and second and third trimester normotensives did not. Deglycosylating plasma proteins significantly increased adhesion in all the cohorts. These results support a role of plasma glycoprotein interaction in monocyte-endothelial cell adhesion and could suggest a novel therapeutic target for severe preeclampsia.

Elevated ratio of maternal plasma ApoCIII to ApoCII in preeclampsia.

Preeclampsia is a hypertensive disorder unique to pregnancy. Although the pathogenesis of the disease begins with aberrant spiral artery invasion in the first trimester, clinical symptoms usually do not present until late in pregnancy. Apolipoprotein CII (ApoCII) and its negative regulator, apolipoprotein CIII (ApoCIII), have recently been described as atherogenesis biomarkers in models of cardiovascular disease. Given the similarities in pathology, etiology, and clinical presentation between cardiovascular disease and preeclampsia, we hypothesized that the ratio of ApoCIII to ApoCII in maternal first trimester plasma would predict preeclampsia later in pregnancy. To test this hypothesis, plasma was prospectively collected from 311 nulliparas at 8 to 12 weeks gestation. After delivery, patients were divided into cohorts based on preeclampsia diagnosis. Conditioning monocytes with preeclamptic plasma potentiated monocyte adhesion to endothelial cells in an in vitro model. The ratio of ApoCIII to ApoCII was significantly elevated in patients with severe preeclampsia relative to normotensive and gestational hypertensive individuals (P < .05) as determined by mass spectrometry and competitive enzyme-linked immunosorbent assay (ELISA) assays. These results support a predictive change in the ratio of ApoCIII to ApoCII in pregnancies complicated by severe preeclampsia.

Microsatellite loci within sixgill sharks, Hexanchus griseus.

Novel sixgill shark (Hexanchus griseus) microsatellite loci were developed and tested on five shark species. A suite of microsatellite loci previously developed for lemon sharks (Negaprion brevirostris) was also tested. Data on 15 microsatellites are presented including primer sequences, number of alleles (a), observed (H(O) ) and expected heterozygosities (H(E) ), and F(IS) values for sixgill sharks (a = 10-69, H(O)  = 0.24-1.00, H(E)  = 0.76-0.96 and F(IS)  = -0.21-0.60), sevengill sharks (Notorynchus cepedianus) (a = 6-40, H(O)  = 0.20-0.73, H(E)  = 0.59-0.94 and F(IS)  = -0.47-0.58), Pacific spiny dogfish (Squalus acanthias) (a = 3-13, H(O)  = 0.00-0.96, H(E)  = 0.24-0.93 and F(IS)  =-0.52-1.00), angle sharks (Squatina californica) (a = 1-4, H(O)  = 0.00-1.00, H(E)  = 0.60-1.00 and F(IS)  =-1.00-0.25), and leopard sharks (Triakis semifasciata) (a = 3-16, H(O)  = 0.20-1.00, H(E)  = 0.53-0.92 and F(IS)  = -0.57-1.00). A final suite of 14 microsatellites (13 developed from sixgill sharks and one from lemon sharks) were found to be polymorphic and conform to Hardy-Weinberg equilibrium within sixgill sharks.

A BAC library of the East African haplochromine cichlid fish Astatotilapia burtoni.

A BAC library was constructed from Astatotilapia burtoni, a haplochromine cichlid that is found in Lake Tanganyika, East Africa, and its surrounding rivers. The library was generated from genomic DNA of blood cells and comprises 96,768 individual clones. Its median insert size is 150 kb and the coverage is expected to represent about 14 genome equivalents. The coverage evaluation was based on genome size estimates that were obtained by flow cytometry. In addition, hybridization screens with five probes largely corroborate the above coverage estimate, although the number of clones ranged from 5 to 22 authenticated clones per single copy probe. The BAC library described here is expected to be useful to the scientific community interested in cichlid genomics as an important resource to gain new insights into the rapid evolution of the great species diversity of haplochromine cichlid fishes.

Ancient divergence of a complex family of immune-type receptor genes.

Multigene families of activating/inhibitory receptors belonging to the immunoglobulin superfamily (IgSF) regulate immunological and other cell-cell interactions. A new family of such genes, termed modular domain immune-type receptors (MDIRs), has been identified in the clearnose skate (Raja eglanteria), a phylogenetically ancient vertebrate. At least five different major forms of predicted MDIR proteins are comprised of four different subfamilies of IgSF ectodomains of the intermediate (I)- or C2-set. The predicted number of individual IgSF ectodomains in MDIRs varies from one to six. MDIR1 contains a positively charged transmembrane residue and MDIR2 and MDIR3 each possesses at least one immunoreceptor tyrosine-based inhibitory motif in their cytoplasmic regions. MDIR4 and MDIR5 lack characteristic activating/inhibitory signalling motifs. MDIRs are encoded in a particularly large and complex multigene family. MDIR domains exhibit distant sequence similarity to mammalian CMRF-35-like molecules, polymeric immunoglobulin receptors, triggering receptors expressed on myeloid cells (TREMs), TREM-like transcripts, NKp44 and FcR homologs, as well as to sequences identified in several different vertebrate genomes. Phylogenetic analyses suggest that MDIRs are representative members of an extended family of IgSF genes that diverged before or very early in evolution of the vertebrates and subsequently came to occupy multiple, fully independent distributions in the present day.