Exploring the potential role of CRTH2 agonists in the prevention of inflammation induced preterm labour

Introduction: Preterm labour occurs in 76,000 women in England and Wales each year and accounts for 75% of neonatal mortality. Infection and inflammation are key triggers of preterm labour. The transcription factor NF-ĸB plays a key regulatory role in the expression of the labour associated genes such as COX-2, prostaglandins and MMPs, as well as pro-inflammatory cytokines such as IL-8, IL-1β, IFN-γ and TNF-α. The anti-inflammatory prostaglandin 15-deoxyΔ-12,14 Prostaglandin J2, a CRTH2 agonist can inhibit NF-ĸB and delay preterm labour in a murine model of inflammation induced preterm labour. It is well recognised that the immunology of pregnancy is biased towards a Th2 rather than Th1 response. CRTH2 agonists induce the production of the anti-inflammatory cytokines IL-4 and IL-10 in T-helper 2 cells in vitro, thus have potential to further alter the Th1:Th2 ratio in favour of the Th2 bias. Aims: This thesis examines the role of the CRTH2 receptor and its agonists in pregnancy and parturition, and its potential as a therapeutic target for the prevention of preterm labour. Methods: PCR, western analysis and flow cytometry were used for CRTH2 expression studies. To examine the effect of CRTH2 agonists on NF-ĸB activation, western analysis of p65 and phospho-p65 was performed. The effect of the agonists on interleukin profiles was demonstrated by flow cytometry. Myometrial contractility response to agonists was measured using a myograph. To determine the in vivo effect of a small molecule CRTH2 agonist on inflammation induced preterm labour, CD1 mice were given an intrauterine injection of LPS and the CRTH2 agonist. Delivery outcome and the effect on labour associated genes and interleukins were determined by PCR, western analysis and ELISA. Results: Amniocytes and myocytes show low CRTH2 mRNA levels however do not express the protein at detectable levels. The small molecule CRTH2 agonist Pyl A does not inhibit NF-ĸB in amniocytes and myocytes, indicating that the inhibition of NF-ĸB by 15dPGJ2 is independent of CRTH2. Although 15dPGJ2 was able to inhibit the production of pro-inflammatory cytokines in peripheral blood mononuclear cells, Pyl A was unable to alter the Th2 cytokine profile. Pyl A inhibits myometrial contractility, although in a mechanism independent of CRTH2. In the murine model of inflammation induced preterm labour fetal viability was significantly increased by Pyl A, however, preterm labour was augmented rather than prevented. Conclusion: 15dPGJ2 inhibits NF-ĸB and inflammation induced preterm labour in the mouse in a mechanism independent of CRTH2. Contrary to anticipated, Pyl A does not modulate the Th1:Th2 bias or delay preterm labour in vivo. However, 15dPGJ2 suppresses the Th1 response in vitro, thus represents a potential anti-inflammatory therapeutic target for both the prevention of preterm labour and inflammation induced brain injury

The Th1:Th2 Dichotomy of Pregnancy and Preterm Labour

Pregnancy is a unique immunological state in which a balance of immune tolerance and suppression is needed to protect the fetus without compromising the mother. It has long been established that a bias from the T helper 1 cytokine profile towards the T helper 2 profile contributes towards successful pregnancy maintenance. The majority of publications that report on aberrant Th1:Th2 balance focus on early pregnancy loss and preeclampsia. Over the last few decades, there has been an increased awareness of the role of infection and inflammation in preterm labour, and the search for new biomarkers to predict preterm labour continues. In this paper, we explore the evidence for an aberrant Th1:Th2 profile associated with preterm labour. We also consider the potential for its use in screening women at high risk of preterm labour and for prophylactic therapeutic measures for the prevention of preterm labour and associated neonatal adverse outcomes

Changes in the Th1 : Th2 Cytokine Bias in Pregnancy and the Effects of the Anti-Inflammatory Cyclopentenone Prostaglandin 15-Deoxy-Δ12,14-Prostaglandin J2

Pregnancy is a complex immunological state in which a bias towards T helper 2 (Th2) protects the fetus. Evidence suggests that proinflammatory cytokines increase the risk of poor neonatal outcome, independently of the direct effect of preterm labour. The anti-inflammatory prostaglandin 15-deoxy-Δ12,14-Prostaglandin J2 (15dPGJ2) inhibits nuclear factor Kappa B (NF-κB) in amniocytes and myocytes in vitro and is a ligand for the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptor. Here we examine the Th1:Th2 cytokine bias in pregnancy and whether 15dPGJ2 could be used to inhibit the production of the proinflammatory cytokines through inhibition of NF-κB while simultaneously promoting Th2 interleukin 4 (IL-4) synthesis via CRTH2 in T helper cells. Peripheral blood mononuclear cells (PBMCs) from women at 28 weeks, term pre-labour, term labour as well as non-pregnant female controls were cultured with 15dPGJ2 or vehicle control and stimulated with phorbol myristyl acetate (PMA)/ionomycin. The percentage of CD4+ cells producing interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in response to PMA/ionomycin was significantly reduced in pregnancy. 15dPGJ2 reduced IFN-γ and TNF-α production in stimulated T helper cells, but did not alter IL-4 production in CRTH2+ve cells. 15dPGJ2 also reduced phospho-p65 in stimulated PBMCs. In summary, 15dPGJ2 suppresses the Th1 response of PBMCs during pregnancy and active labour whilst maintaining the Th2 response suggesting a therapeutic benefit in reducing neonatal morbidity in inflammation-induced PTL

Prostaglandin F2α requires activation of calcium-dependent signalling to trigger inflammation in human myometrium

IntroductionPreterm birth is one of the major causes of neonatal morbidity and mortality across the world. Both term and preterm labour are preceded by inflammatory activation in uterine tissues. This includes increased leukocyte infiltration, and subsequent increase in chemokine and cytokine levels, activation of pro-inflammatory transcription factors as NF-κB and increased prostaglandin synthesis. Prostaglandin F2α (PGF2α) is one of the myometrial activators and stimulators.MethodsHere we investigated the role of PGF2α in pro-inflammatory signalling pathways in human myometrial cells isolated from term non-labouring uterine tissue. Primary myometrial cells were treated with G protein inhibitors, calcium chelators and/or PGF2α. Nuclear extracts were analysed by TranSignal cAMP/Calcium Protein/DNA Array. Whole cell protein lysates were analysed by Western blotting. mRNA levels of target genes were analysed by RT-PCR.ResultsThe results show that PGF2α increases inflammation in myometrial cells through increased activation of NF-κB and MAP kinases and increased expression of COX-2. PGF2α was found to activate several calcium/cAMP-dependent transcription factors, such as CREB and C/EBP-β. mRNA levels of NF-κB-regulated cytokines and chemokines were also elevated with PGF2α stimulation. We have shown that the increase in PGF2α-mediated COX-2 expression in myometrial cells requires coupling of the FP receptor to both Gαq and Gαi proteins. Additionally, PGF2α-induced calcium response was also mediated through Gαq and Gαi coupling.DiscussionIn summary, our findings suggest that PGF2α-induced inflammation in myometrial cells involves activation of several transcription factors – NF-κB, MAP kinases, CREB and C/EBP-β. Our results indicate that the FP receptor signals via Gαq and Gαi coupling in myometrium. This work provides insight into PGF2α pro-inflammatory signalling in term myometrium prior to the onset of labour and suggests that PGF2α signalling pathways could be a potential target for management of preterm labour

Implementation of clinical decision support to manage acute kidney injury in secondary care: an ethnographic study

Background: Over the past decade, acute kidney injury (AKI) has become a global priority for improving patient safety and health outcomes. In the UK, a confidential inquiry into AKI led to the publication of clinical guidance and a range of policy initiatives. National patient safety directives have focused on the mandatory establishment of clinical decision support systems (CDSSs) within all acute National Health Service (NHS) trusts to improve the detection, alerting and response to AKI. We studied the organisational work of implementing the AKI CDSSs within routine hospital care. Methods: An ethnographic study comprising non-participant observation and interviews was conducted in two NHS hospitals, delivering AKI quality improvement programmes, located in one region of England. Three researchers conducted a total of 49 interviews and 150 hours of observation over an 18-month period. Analysis was conducted collaboratively and iteratively around emergent themes, relating to the organisational work of technology adoption. Results: The two hospitals developed and implemented AKI CDSSs using very different approaches. Nevertheless, both resulted in adaptive work and trade-offs relating to the technology, the users, the organisation, and the wider system of care. A common tension was associated with attempts to maximise benefit whilst minimise additional burden. In both hospitals, resource pressures exacerbated the tensions of translating AKI recommendations into routine practice. Conclusions Our analysis highlights a conflicted relationship between external context (policy and resources), and organisational structure and culture (e.g. digital capability, attitudes to quality improvement). Greater consideration is required to the long-term effectiveness of the approaches taken, particularly in light of the ongoing need for adaptation to incorporate new practices into routine work

Chemoattractant Receptor Homologous to the T Helper 2 Cell (CRTH2) Is Not Expressed in Human Amniocytes and Myocytes

BACKGROUND: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. METHODS: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. RESULTS: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Strict evolutionary conservation followed rapid gene loss on human and rhesus Y chromosomes

The human X and Y chromosomes evolved from an ordinary pair of autosomes during the past 200–300 million years[superscript 1, 2, 3]. The human MSY (male-specific region of Y chromosome) retains only three percent of the ancestral autosomes’ genes owing to genetic decay[superscript 4, 5]. This evolutionary decay was driven by a series of five ‘stratification’ events. Each event suppressed X–Y crossing over within a chromosome segment or ‘stratum’, incorporated that segment into the MSY and subjected its genes to the erosive forces that attend the absence of crossing over[superscript 2, 6]. The last of these events occurred 30 million years ago, 5 million years before the human and Old World monkey lineages diverged. Although speculation abounds regarding ongoing decay and looming extinction of the human Y chromosome[superscript 7, 8, 9, 10], remarkably little is known about how many MSY genes were lost in the human lineage in the 25 million years that have followed its separation from the Old World monkey lineage. To investigate this question, we sequenced the MSY of the rhesus macaque, an Old World monkey, and compared it to the human MSY. We discovered that during the last 25 million years MSY gene loss in the human lineage was limited to the youngest stratum (stratum 5), which comprises three percent of the human MSY. In the older strata, which collectively comprise the bulk of the human MSY, gene loss evidently ceased more than 25 million years ago. Likewise, the rhesus MSY has not lost any older genes (from strata 1–4) during the past 25 million years, despite its major structural differences to the human MSY. The rhesus MSY is simpler, with few amplified gene families or palindromes that might enable intrachromosomal recombination and repair. We present an empirical reconstruction of human MSY evolution in which each stratum transitioned from rapid, exponential loss of ancestral genes to strict conservation through purifying selection