Epigenetic regulation of glucose transporters in non-small cell lung cancer

Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs.

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus.

Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low-stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules.

The expression and regulation of pregnancy-specific glycoproteins in the mouse

Pregnancy-Specific Glycoproteins (PSG) are the most abundant fetally expressed proteins in the maternal bloodstream at term. This multigene family are immunoglobulin superfamily members and are predominantly expressed in the syncytiotrophoblast of human placenta and in giant cells and spongiotrophoblast of rodent placenta. PSGs are encoded by seventeen genes in the mouse and ten genes in the human. Little is known about the function of this gene family, although they have been implicated in immune modulation and angiogenesis through the induction of cytokines such as IL-10 and TGFβ1 in monocytes, and more recently, have been shown to inhibit the platelet-fibrinogen interaction. I provide new information concerning the evolution of the murine Psg genomic locus structure and organisation, through the discovery of a recent gene inversion event of Psg22 within the major murine Psg cluster. In addition to this, I have performed an examination of the expression patterns of individual Psg genes in placental and non-placental tissues. This study centres on Psg22, which is the most abundant murine Psg transcript detected in the first half of pregnancy. A novel alternative splice variant transcript of Psg22 lacking the protein N1-domain was discovered, and similar to the full length isoform induces TGFβ1 in macrophage and monocytic cell lines. The identification of a bidirectional antisense long non-coding RNA transcript directly adjacent to Psg22 and its associated active local chromatin conformation, suggests an interesting epigenetic gene-specific regulatory mechanism that may be responsible for the high level of Psg22 expression relative to the other Psg family members upon trophoblast giant cell differentiation

A review and psychometric evaluation of pregnancy-specific stress measures

Considerable evidence has accumulated on the association between pregnancy-specific stress and adverse birth outcomes with an increasing number of measures of pregnancy-specific stress being developed internationally. However, the introduction of these measures has not always been theoretically or psychometrically grounded, resulting in questions about the quality and direction of such research. This review summarizes evidence on the reliability and validity of pregnancy-specific stress measures identified between 1980 and October 2010. Fifteen pregnancy-specific stress measures were identified. Cronbach’s alpha coefficient ranged from 0.51–0.96 and predictive validity data on preterm birth were reported for five measures. Convergent validity data suggest that pregnancy-specific stress is related to, but distinct from, global stress. Findings from this review consolidate current knowledge on pregnancy-specific stress as a consistent predictor of premature birth. This review also advances awareness of the range of measures of pregnancy-specific stress and documents their strengths and limitations based on published reliability and validity data. Careful consideration needs to be given as to which measures to use in future research to maximize the development of stress theory in pregnancy and appropriate interventions for women who experience stress in pregnancy. An international, strategic collaboration is recommended to advance knowledge in this area of study.

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H

Although the importance of CD4(+) T cell responses to human cytonnegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4(+) CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HICMV isolates revealed that the gB and gH epitope sequences recognized by human CD4(+) T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4(+) CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse pepticle-sensitized Patr-DR7(+) cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class 11 lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.

Structural basis for the platelet-collagen interaction - The smallest motif within collagen that recognizes and activates platelet glycoprotein VI contains two glycine-proline-hydroxyproline triplets

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)(n) backbone. The adhesion of recombinant human Glycoprotein VI ectodomain, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO center dot center dot center dot center dot center dot center dot center dot center dot center dot GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)(4) motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.

Fetal Movements in late Pregnancy : Categorization, Self-assessment, and Prenatal Attachment in relation to women’s experiences

Aim: To explore how pregnant women experience fetal movements in late pregnancy. Specific aims were:  to study women’s experiences during the time prior to receiving news that their unborn baby had died in utero (I), to investigate women’s descriptions of fetal movements (II), investigate the association between the magnitude of fetal movements and level of prenatal attachment (III), and to study women’s experiences using two different self-assessment methods (IV). Methods: Interviews, questionnaires, and observations were used. Results: Premonition that something had happened to their unborn baby, based on a lack of fetal movements, was experienced by the participants. The overall theme “something is wrong” describes the women’s insight that the baby’s life was threatened (I). Fetal movements that were sorted into the domain “powerful movements” were perceived in late pregnancy by 96 % of the participants (II). Perceiving frequent fetal movements on at least three occasions per 24 hours was associated with higher scores of prenatal attachment in all the three subscales on PAI-R. The majority (55%) of the 456 participants reported average occasions of frequent fetal movements, 26% several occasions and 18% reported few occasions of frequent fetal movements, during the current gestational week.  (III). Only one of the 40 participants did not find at least one method for monitoring fetal movements suitable. Fifteen of the 39 participants reported a preference for the mindfetalness method and five for the count-to-ten method. The women described the observation of the movements as a safe and reassuring moment for communication with their unborn baby (IV). Conclusion:  In full-term and uncomplicated pregnancies, women usually perceive fetal movements as powerful. Furthermore, women in late pregnancy who reported frequent fetal movements on several occasions during a 24-hour period seem to have a high level of prenatal attachment. Women who used self-assessment methods for monitoring fetal movements felt calm and relaxed when observing the movements of their babies. They had a high compliance for both self-assessment methods. Women that had experienced a stillbirth in late pregnancy described that they had a premonition before they were told that their baby had died in utero. 

Preferences for a third-trimester ultrasound scan in a low-risk obstetric population: a discrete choice experiment

Objective: Establish maternal preferences for a third-trimester ultrasound scan in a healthy, low-risk pregnant population.

Design: Cross-sectional study incorporating a discrete choice experiment.

Setting: A large, urban maternity hospital in Northern Ireland.

Participants: One hundred and forty-six women in their second trimester of pregnancy.

Methods: A discrete choice experiment was designed to elicit preferences for four attributes of a third-trimester ultrasound scan: health-care professional conducting the scan, detection rate for abnormal foetal growth, provision of non-medical information, cost. Additional data collected included age, marital status, socio-economic status, obstetric history, pregnancy-specific stress levels, perceived health and whether pregnancy was planned. Analysis was undertaken using a mixed logit model with interaction effects.

Main outcome measures: Women's preferences for, and trade-offs between, the attributes of a hypothetical scan and indirect willingness-to-pay estimates.

Results: Women had significant positive preference for higher rate of detection, lower cost and provision of non-medical information, with no significant value placed on scan operator. Interaction effects revealed subgroups that valued the scan most: women experiencing their first pregnancy, women reporting higher levels of stress, an adverse obstetric history and older women.

Conclusions: Women were able to trade on aspects of care and place relative importance on clinical, non-clinical outcomes and processes of service delivery, thus highlighting the potential of using health utilities in the development of services from a clinical, economic and social perspective. Specifically, maternal preferences exhibited provide valuable information for designing a randomized trial of effectiveness and insight for clinical and policy decision makers to inform woman-centred care.

The impact of an educational DVD on pregnant women with gestational diabetes mellitus: a randomised controlled trial

Background The diagnosis of gestational diabetes (GDM) during pregnancy can lead to anxiety. Little research has focused on the education these women receive and how this is best delivered in a busy clinic. Aim This study evaluated the impact of an innovative patient-centred educational DVD on anxiety and glycaemic control and in newly diagnosed women with GDM. Method 150 multi-ethnic women, aged 19-44 years, from three UK hospitals were randomised to either standard care plus DVD (DVD group, n=77) or standard care alone (control group, n=73) at GDM diagnosis. Women were followed up at their next clinic visit at a mean ± SD of 2.5 ± 1.6 weeks later. Primary outcomes were anxiety (State-Trait Anxiety Inventory) and mean 1-hour postprandial capillary self-monitored blood glucose for all meals, on day prior to follow-up. Secondary outcomes included pregnancy specific stress (Pregnancy Distress Questionnaire), emotional adjustment to diabetes (Appraisal of Diabetes Scale), self-efficacy (Diabetes Empowerment Scale) and GDM knowledge (non-validated questionnaire). Other outcomes included mean fasting and 1-hour postprandial blood glucose at each meal, on day prior to follow-up. Women in the DVD group completed a feedback questionnaire on the resource. Results No significant difference between the DVD and control group were reported, for anxiety (37.7 ± 11.7 vs 36.2 ± 10.9; mean difference after adjustment for covariates (95%CI) 2.5 (-0.8, 5.9) or for mean 1-hour postprandial glucose (6.9 ± 0.9 vs 7.0 ± 1.2 mmol/L; -0.2 (-0.5, 0.2). Similarly, no significant differences in the other psychosocial variables were identified between the groups. However, the DVD group had significantly lower postprandial breakfast glucose compared to the control group (6.8 ± 1.2 vs 7.4 ± 1.9 mmol/L; -0.5 (-1.1, -<0.1; p=0.04). Using a scale of 0-10, 84% rated the DVD 7 or above for usefulness (10 being very useful), and 88% rated it 7 or above when asked if they would recommend to a friend (10 being very strongly recommend). Women described the DVD as ‘reassuring’, ‘a fantastic tool’, that ‘provided a lot of information in a quick and easy way’ and ‘helped reinforce all the information from clinic’. Discussion While no significant change was observed in anxiety or mean postprandial glucose, the DVD was rated highly by women with GDM and may be a useful resource to assist with educating newly diagnosed women. This project is supported by BRIDGES, an IDF programme supported by an educational grant from Lilly Diabetes.

Morphological and functional characterization of placenta during gestation in bovine clones derived by somatic nuclear transfer

La technique de clonage par transfert nucléaire de cellules somatiques (SCNT) présente une page importante dans les annales scientifiques, mais son application pratique demeure incertaine dû à son faible taux de succès. Les anomalies placentaires et de développement fœtal se traduisent par des pertes importantes de gestation et des mortalités néonatales. Dans un premier temps, la présente étude a caractérisé les changements morphologiques des membranes fœtales durant la gestation clonée en les comparant à des gestations contrôles obtenues à partir de l’insémination artificielle. Les différentes anomalies morphologiques des placentomes telles que l’œdème chorioallantoique, la présence de zones hyperéchoiques et irrégulières dans la membrane amniotique et la présence de cellules inflammatoires dégénérées compromettent le développement fœtal normal de la gestation clonée. L’examen ultrasonographique représente une technique diagnostique importante pour faire le suivi d’une gestation et de caractériser les changements placentaires dans le cadre d’évaluation globale du bien-être fœtal. Le profil hormonal de trois stéroïdes (progestérone (P4), estrone sulfate (E1S), et œstradiol (E2)) et de la protéine B spécifique de gestation (PSPB) dans le sérum des vaches porteuses de clones SCNT a été déterminé et associé aux anomalies de gestations clonées. Une diminution de la P4 sérique au jour 80, une élévation du niveau de la concentration de la PSPB au jour 150, et une augmentation de la concentration d’E2 sérique durant le deuxième et troisième tiers de la gestation clonée coïncident avec les anomalies de gestation déjà reportées. Ces changements du profil hormonal associés aux anomalies phénotypiques du placenta compromettent le déroulement normal de la gestation clonée et gênent le développement et le bien-être fœtal. Sur la base des observations faites sur le placenta de gestation clonée, le mécanisme moléculaire pouvant expliquer la disparition de l’épithélium du placenta (l’interface entre le tissue maternel et le placenta) a été étudié. L’étude a identifié des changements dans l’expression de deux protéines d’adhérence (E-cadhérin et β-catenin) de cellules épithéliales pouvant être associées aux anomalies du placenta chez les gestations clonées. Le tissu de cotylédons provenant de gestations clonées et contrôles a été analysé par Western blot, RT-PCR quantitatif, et par immunohistochimie. Les résultats présentaient une diminution significative (p<0.05) de l’expression des dites protéines dans les cellules trophoblastiques chez les gestations clonées. Le RT-PCR quantitatif démontrait que les gènes CCND1, CLDN1 et MSX1 ciblés par la voie de signalisation de la Wnt/β-catenin étaient significativement sous exprimés. La diminution de l’expression des protéines E-cadherin et β-catenin avec une réduction de l’activation de la protéine β-catenin durant le période d’attachement de l’embryon peut potentiellement expliquer l’absence totale ou partielle de l’attachement des membranes fœtales au tissu maternel et éventuellement, l’insuffisance placentaire caractéristique des gestations clonées chez la vache. La caractérisation morphologique et fonctionnelle du placenta durant les gestations clonées à haut risque est essentielle pour évaluer le statut de la gestation. Les résultats de la présente étude permettront de prédire le développement et le bien-être fœtal de façon critique à travers un protocole standardisé et permettre des interventions médicales pour améliorer le taux de succès des gestations clonées chez les bovins.

Therapeutic patents for the treatment of pre-eclampsia

Pre-eclampsia (PE) is a pregnancy-specific syndrome that is a principal cause of maternal morbidity and mortality, accounting for almost 15% of pregnancy-associated deaths. In its mild form, PE most commonly presents with the features of maternal hypertension and proteinuria but can swiftly and unpredictably become severe with many extensive and life-threatening complications. The diverse symptoms of PE have made it a difficult disease not only to define, but also to identify a causative agent for the symptoms. It has therefore proved difficult to develop specific drugs that can be used to manage the condition. This review examines the patent literature to reveal current findings that exhibit the potential to target the effects of PE with the aim of either preventing or altering the course of this life-threatening disease of pregnancy.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.

A path model of psychosocial and health behaviour change predictors of excessive gestational weight gain

This study aimed to evaluate a conceptual model of psychosocial, behaviour change, and behavioural predictors of excessive gestational weight gain (GWG). Background: Excessive GWG can place women and their babies at risk of poor health outcomes, including obesity. Models of psychosocial and behaviour change predictors of excessive GWG have not been extensively explored; understanding the mechanisms leading to excess GWG will provide crucial evidence towards the development of effective interventions. Method: Two hundred and eighty-eight pregnant women (≤18 weeks gestation) were recruited to a prospective study. Demographic, psychosocial, health behaviour change, and behavioural factors were assessed at 17 (Time 1, T1) and 33 weeks (Time 2, T2) gestation. Pre-pregnancy and final pregnancy weight were obtained and women were classified with/without excessive GWG. Logistic regressions refined the list of predictors of excessive GWG; variables with p < .1 were included in a path analysis. Results: Age, family income, T2 depression, T2 pregnancy-specific coping, T1 buttocks dissatisfaction, T2 GWG-specific self-efficacy, T1 dietary readiness, T1 dietary importance, and T1 vegetable intake predicted excessive GWG in the logistic regressions and were included in the path model. The baseline path model demonstrated poor fit. Once statistically and theoretically plausible paths were added, adequate model fit was achieved (χ² = 21.61(9), p < .05; RMSEA = .07; CFI = .93); this revised model explained 19.5% of the variance in excessive GWG. Women with high T1 buttocks dissatisfaction were more likely to exhibit low levels of dietary readiness. Women with low dietary readiness were more likely to have a lower vegetable intake, which predicted excessive GWG. Women with higher T2 depressive symptoms were more likely to report lower GWG self-efficacy and gain excessively. Conclusion: Future behavioural GWG trials should consider combining psychosocial and health behaviour change factors to optimise GWG.