Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 X 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. Al; the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN 10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn 10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

GABA(A) receptor sites in the developing human foetus

GABA(A) receptor sites were characterised in cerebral cortex tissue samples from deceased neurologically normal infants who had come to autopsy during the third trimester of pregnancy. Pharmacological parameters were obtained from homogenate binding studies which utilised the 'central-type' benzodiazepine ligands [H-3]diazepam and [H-3]flunitrazepam, and from the GABA activation of [H-3]diazepam binding. It was found that the two radioligands behaved differently during development. The affinity of [H-3]flunitrazepam for its binding site did not vary significantly between preparations, whereas the [H-3]diazepam K-D showed marked regional and developmental variations: infant tissues showed a distinctly lower affinity than adults for this ligand. The density of [H-3]flunitrazepam binding sites increased similar to35% during the third trimester to reach adult levels by term, whereas [H-3]diazepam binding capacity declined slightly but steadily throughout development. The GABA activation of [H-3]diazepam binding was less efficient early in the trimester, in that the affinity of the agonist was significantly lower, though it rose to adult levels by term. The strength of the enhancement response increased to adult levels over the same time-frame. The results strongly suggest that the subunit composition of cortical GABA(A) sites changes significantly during this important developmental stage. (C) 2002 Elsevier Science B.V. All rights reserved.

A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: Total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH2S)(28-29,56-57,76-77)]

The 101 residue protein early pregnancy factor (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH2CH2SH thiolate with XCH2CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH2CH2SH thiolate with a BrCH2CO-peptide than with a CICH2CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N-alpha-chloroacetyl, C-terminal thiolated peptide with a second N-alpha-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N-alpha-chloroacetyl peptides could be activated by halide exchange using saturated KI solutions to yield the highly reactive No-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or cassettes comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH2CH2SH] 13, ClAc-[EPF 30-55]-[NHCH2CH2SH] 14, and Ac-[EPF 1-27]-[NHCH2CH2SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH2S)(28-29,56-57,76-77)) 19 in 19% overall yield.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Effect of oxidative stress upon absorption of glucose by the human placenta: in vitro studies with BeWo cells

Pregnancy is a dynamic state and the placenta is a temporary organ that, among other important functions, plays a crucial role in the transport of nutrients and metabolites between the mother and the fetus, which is essential for a successful pregnancy. Among these nutrients, glucose is considered a primary source of energy and, therefore, fundamental to insure proper fetus development. Several studies have shown that glucose uptake is dependent on several morphological and biochemical placental conditions. Oxidative stress results from the unbalance between reactive oxygen species (ROS) and antioxidants, in favor of the first. During pregnancy, ROS, and therefore oxidative stress, increase, due to increased tissue oxygenation. Moreover, the relation between ROS and some pathological conditions during pregnancy has been well established. For these reasons, it becomes essential to understand if oxidative stress can compromise the uptake of glucose by the placenta. To make this study possible, a trophoblastic cell line, the BeWo cell line, was used. Experiments regarding glucose uptake, either under normal or oxidative stress conditions, were conducted using tert-butylhydroperoxide (tBOOH) as an oxidative stress inducer, and 3H-2-deoxy-D-glucose (3H-DG) as a glucose analogue. Afterwards, studies regarding the involvement of glucose facilitative transporters (GLUT) and the phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC) pathways were conducted, also under normal and oxidative stress conditions. A few antioxidants, endogenous and from diet, were also tested in order to study their possible reversible effect of the oxidative effect of tBOOH upon apical 3H-DG uptake. Finally, transepithelial studies gave interesting insights regarding the apical-to-basolateral transport of 3H-DG. Results showed that 3H-DG uptake, in BeWo cells, is roughly 50% GLUT-mediated and that tBOOH (100 μM; 24h) decreases apical 3H-DG uptake in BeWo cells by about 33%, by reducing both GLUT- (by 28%) and non-GLUT-mediated (by 40%) 3H-DG uptake. Uptake of 3H-DG and the effect of tBOOH upon 3H-DG uptake are not dependent on PKC and PI3K. Moreover, the effect of tBOOH is not associated with a reduction in GLUT1 mRNA levels. Resveratrol, quercetin and epigallocatechin-3-gallate, at 50 μM, reversed, by at least 45%, the effect of tBOOH upon 3H-DG uptake. Transwell studies show that the apical-to-basolateral transepithelial transport of 3H-DG is increased by tBOOH.In conclusion, our results show that tBOOH caused a marked decrease in both GLUT and non-GLUT-mediated apical uptake of 3H-DG by BeWo cells. Given the association of increased oxidative stress levels with several important pregnancy pathologies, and the important role of glucose for fetal development, the results of this study appear very interesting.

Label-free human chorionic gonadotropin detection at picogram levels using oriented antibodies bound to graphene screen-printed electrodes

Human chorionic gonadotropin (hCG) is a key diagnostic marker of pregnancy and an important biomarker for cancers in the prostate, ovaries and bladder and therefore of great importance in diagnosis. For this purpose, a new immunosensor of screen-printed electrodes (SPEs) is presented here. The device was fabricated by introducing a polyaniline (PANI) conductive layer, via in situ electropolymerization of aniline, onto a screen-printed graphene support. The PANI-coated graphene acts as the working electrode of a three terminal electrochemical sensor. The working electrode is functionalised with anti-hCG, by means of a simple process that enabled oriented antibody binding to the PANI layer. The antibody was attached to PANI following activation of the –COOH group at the Fc terminal. Functionalisation of the electrode was analysed and optimized using Electrochemical Impedance Spectroscopy (EIS). Chemical modification of the surface was characterised using Fourier transform infrared, and Raman spectroscopy with confocal microscopy. The graphene–SPE–PANI devices displayed linear responses to hCG in EIS assays from 0.001 to 50 ng mL−1 in real urine, with a detection limit of 0.286 pg mL−1. High selectivity was observed with respect to the presence of the constituent components of urine (urea, creatinine, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, ammonium chloride, potassium sulphate and sodium chloride) at their normal levels, with a negligible sensor response to these chemicals. Successful detection of hCG was also achieved in spiked samples of real urine from a pregnant woman. The immunosensor developed is a promising tool for point-of-care detection of hCG, due to its excellent detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Immunological response in cases of complicated and uncomplicated bartonellosis during pregnancy

Bartonellosis (Carrion's Disease) during pregnancy is associated with high rates of maternal and perinatal mortality. We report the immunological patterns in two cases of human bartonellosis during pregnancy. One patient had an uncomplicated course while the second patient developed life threatening anasarca and cardiac tamponade. The patient with a complicated course had a Th1 response with a higher elevation of IL-10. This elevation has been associated with poor outcome pregnancies during bacterial infections.

Burkitt’s Lymphoma Related to Epstein–Barr Virus Infection During Pregnancy

Setting: Burkitt’s lymphoma is a rare form of cancer and is an extremely rare diagnosis during pregnancy. This form of lymphoma is a very fast growing B cell neoplasm and chemotherapy is the treatment of choice for the disease in all its stages. Case report: The authors describe the case of a Caucasian 40-year-old nulliparous woman, with previous known Epstein–Barr virus infection, that presents at 28 weeks gestation with supraclavicular adenopathy and multiple bilateral breast nodules, in which biopsy showed non-Hodgkin lymphoma, Burkitt’s type. Discussion: There are few described cases of Burkitt’s lymphoma during pregnancy and in general the outcomes have been poor. In most of the cases, the patients were not treated by current standards or instead had a late diagnosis. This neoplasia is the most rapidly progressive human tumor, and any delay in initiating therapy can adversely aVect patient’s prognosis. The authors discuss treatment options in pregnancy and its perinatal implications.

Vertical transmission of Cryptococcus neoformans from a mother coinfected with human immunodeficiency virus: case report

Disseminated infection with Cryptococcus neoformans was observed in a newborn infant who presented fever and respiratory symptoms since the 52nd day of life. The mother was infected by human immunodeficiency virus and presented pulmonary and meningeal cryptococcal infection. This is a rare case of cryptococcal infection with probable maternal-fetal transmission.

Unraveling maternal and fetal genetic factors protecting from Pregnancy Associated Malaria in the mouse

Malaria is one of the most devastating diseases in the world. In Plasmodium endemic regions, pregnant women are among the most vulnerable groups. Pregnancy Associated Malaria (PAM) threatens both maternal and foetal lives. Despite differences between human and mouse placentas PAM mouse models recapitulate key pathological features of human PAM. Here we describe new PAM models of mid gestation infection in the C57BL/6 mouse.(...)

Mother's anxiety and depression during the third pregnancy trimester and neonate's mother versus stranger's face/voice visual preference

Background: Neonates show visual preference for their mother's face/voice and shift their attention from their mother to a stranger's face/voice after habituation. Aim: To assess neonate's mother versus stranger's face/voice visual preference, namely mother's anxiety and depression during the third pregnancy trimester and neonate's: 1) visual preference for the mother versus the stranger's face/voice (pretest visual preference), 2) habituation to the mother's face/voice and 3) visual preference for the stranger versus the mother's face/voice (posttest visual preference). Method: Mothers (N=100) filled out the Edinburgh Postnatal Depression Scale (EPDS) and the State Anxiety Inventory (STAI) both at the third pregnancy trimester and childbirth, and the “preference and habituation to the mother's face/voice versus stranger” paradigm was administered to their newborn 1 to 5 days after childbirth. Results: Neonates of anxious/depressed mothers during the third pregnancy trimester contrarily to neonates of non-anxious/non-depressed mothers did not look 1) longer at their mother's than at the stranger's face/voice at the pretest visual preference (showing no visual preference for the mother), nor 2) longer at the stranger's face/voice in the posttest than in the pretest visual preference (not improving their attention to the stranger's after habituation). Conclusion: Infants exposed to mother's anxiety/depression at the third gestational trimester exhibit less perceptual/social competencies at birth.

Intracellular bacteria and adverse pregnancy outcomes.

Clin Microbiol Infect 2011; 17: 1312-1322 ABSTRACT: This review considers the role of intracellular bacteria in adverse pregnancy outcomes, such as miscarriage, stillbirths, and preterm labour. The cause of miscarriage, stillbirth and preterm labour often remains unexplained. Intracellular bacteria that grow either poorly or not at all on media used routinely to detect human pathogens could be the aetiological agents of these obstetric conditions. For example, Listeria monocytogenes and Coxiella burnetti are intracellular bacteria that have a predilection for the fetomaternal unit and may induce fatal disease in the mother and/or fetus. Both are important foodborne or zoonotic pathogens in pregnancy. Preventive measures, diagnostic tools and treatment will be reviewed. Moreover, we will also address the importance in adverse pregnancy outcomes of other intracellular bacteria, including Brucella abortus and various members of the order Chlamydiales. Indeed, there is growing evidence that Chlamydia trachomatis, Chlamydia abortus and Chlamydia pneumoniae infections may also result in adverse pregnancy outcomes in humans and/or animals. Moreover, newly discovered Chlamydia-like organisms have recently emerged as new pathogens of both animals and humans. For example, Waddlia chondrophila, a Chlamydia-related bacterium isolated from aborted bovine fetuses, has also been implicated in human miscarriages. Future research should help us to better understand the pathophysiology of adverse pregnancy outcomes caused by intracellular bacteria and to determine the precise mode of transmission of newly identified bacteria, such as Waddlia and Parachlamydia. These emerging pathogens may represent the tip of the iceberg of a large number of as yet unknown intracellular pathogenic agents.

Protection of Life During Pregnancy Bill

An Act to protect human life during pregnancy; to make provision for reviews at the instigation of a pregnant woman of certain medical opinions given in respect of pregnancy; to provide for an offence of intentional destruction of unborn human life; to amend the Health Act 2007; to repeal sections 58 and 59 of the Offences Against the Person Act 1861; and to provide for matters connected therewith. Click here to download PDF 296KB