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Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. Here we investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Mass spectrometry analysis revealed that the placental O-GlcNAcome is altered in women with type 1 (n = 6) or type 2 (n = 6) diabetes T2D (≥ twofold change in abundance in 162 and 165 GlcNAcylated proteins respectively compared to BMI-matched controls n = 11). Ingenuity pathway analysis indicated changes to clathrin-mediated endocytosis (CME) and CME-associated proteins, clathrin, Transferrin (TF), TF receptor and multiple Rabs, were identified as O-GlcNAcylation targets. Stimulating protein O-GlcNAcylation using glucosamine (2.5 mM) increased the rate of TF endocytosis by human placental cells (p = 0.02) and explants (p = 0.04). Differential GlcNAcylation of CME proteins suggests altered transfer of cargo by placentas of women with pre-gestational diabetes, which may contribute to alterations in fetal growth. The human placental O-GlcNAcome provides a resource to aid further investigation of molecular mechanisms governing placental nutrient sensing.

BackgroundAntimicrobial resistance is a serious global health concern that emphasizes completing treatment course. Recently, the effectiveness of short versus longer antibiotic courses has been questioned. This study investigated the duration of prescribed antibiotics, their effectiveness, and associated risk of infection-related complications.MethodsClinical Practice Research Datalink identified 4 million acute infection episodes prescribed an antibiotic in primary care between January 2014—June 2014, England. Prescriptions were categorized by duration. Risk of infection-related hospitalizations within 30 days was modelled overall and by infection type. Risk was assessed immediately after or within 30 days follow-up to measure confounders given similar and varying exposure, respectively. An interaction term with follow-up time assessed whether hazard ratios (HRs) remained parallel with different antibiotic durations.ResultsThe duration of antibiotic courses increased over the study period (5.2–19.1%); 6–7 days were most common (66.9%). Most infection-related hospitalizations occurred with prescriptions of 8–15 days (0.21%), accompanied by greater risk of infection-related complications compared to patients who received a short prescription (HR: 1.75 [95% CI: 1.54–2.00]). Comparing HRs in the first 5 days versus remaining follow-up showed longer antibiotic courses were no more effective than shorter courses (1.02 [95% CI: 0.90–1.16] and 0.92 [95% CI: 0.75–1.12]). No variation by infection-type was observed.ConclusionsEqual effectiveness was found between shorter and longer antibiotic courses and the reduction of infection-related hospitalizations. Stewardship programs should recommend shorter courses of antibiotics for acute infections. Further research is required for treating patients with a complex medical history.SummaryPrescribing of longer courses increased over the study period. The majority of hospitalizations occurred for patients receiving longer courses. Risk of developing a complication (immediate vs remaining follow-up) found longer courses were no more effective than shorter courses.

BackgroundThere is a need to reduce antimicrobial uses in humans. Previous studies have found variations in antibiotic (AB) prescribing between practices in primary care. This study assessed variability of AB prescribing between clinicians.MethodsClinical Practice Research Datalink, which collects electronic health records in primary care, was used to select anonymised clinicians providing 500+ consultations during 2012–2017. Eight measures of AB prescribing were assessed, such as overall and incidental AB prescribing, repeat AB courses and extent of risk-based prescribing. Poisson regression models with random effect for clinicians were fitted.Results6111 clinicians from 466 general practices were included. Considerable variability between individual clinicians was found for most AB measures. For example, the rate of AB prescribing varied between 77.4 and 350.3 per 1000 consultations; percentage of repeat AB courses within 30 days ranged from 13.1% to 34.3%; predicted patient risk of hospital admission for infection-related complications in those prescribed AB ranged from 0.03% to 0.32% (5th and 95th percentiles). The adjusted relative rate between clinicians in rates of AB prescribing was 5.23. Weak correlation coefficients (ConclusionsThe wide variation seen in all measures of AB prescribing and weak correlation between them suggests that a single AB measure, such as prescribing rate, is not sufficient to underpin the optimisation of AB prescribing.

Background This study aimed to evaluate the clinical safety of delayed antibiotic prescribing for upper respiratory tract infections (URTIs), which is recommended in treatment guidelines for less severe cases. Methods Two population-based cohort studies used the English Clinical Practice Research Databank and Welsh Secure Anonymized Information Linkage, containing electronic health records from primary care linked to hospital admission records. Patients with URTI and prescriptions of amoxicillin, clarithromycin, doxycycline, erythromycin, or phenoxymethylpenicillin were identified. Patients were stratified according to delayed and immediate prescribing relative to URTI diagnosis. Outcome of interest was infection-related hospital admission after 30 days. Results The population included 1.82 million patients with an URTI and antibiotic prescription; 91.7% had an antibiotic at URTI diagnosis date (immediate) and 8.3% had URTI diagnosis in 1–30 days before (delayed). Delayed antibiotic prescribing was associated with a 52% increased risk of infection-related hospital admissions (adjusted hazard ratio, 1.52; 95% confidence interval, 1.43–1.62). The probability of delayed antibiotic prescribing was unrelated to predicted risks of hospital admission. Analyses of the number needed to harm showed considerable variability across different patient groups (median with delayed antibiotic prescribing, 1357; 2.5% percentile, 295; 97.5% percentile, 3366). Conclusions This is the first large population-based study examining the safety of delayed antibiotic prescribing. Waiting to treat URTI was associated with increased risk of hospital admission, although delayed antibiotic prescribing was used similarly between high- and low-risk patients. There is a need to better target delayed antibiotic prescribing to URTI patients with lower risks of complications., Delayed antibiotic prescribing in patients with upper respiratory tract infections was associated with a 52% increased risk of infection-related hospital admissions in 2 large population-based cohorts. Delayed antibiotic prescribing should be targeted only to patients with lower risks of complications.

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRβ-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

Objectives To examine variations across general practices and factors associated with antibiotic prescribing for common infections in UK primary care to identify potential targets for improvement and optimization of prescribing. Methods Oral antibiotic prescribing for common infections was analysed using anonymized UK primary care electronic health records between 2000 and 2015 using the Clinical Practice Research Datalink (CPRD). The rate of prescribing for each condition was observed over time and mean change points were compared with national guideline updates. Any correlation between the rate of prescribing for each infectious condition was estimated within a practice. Predictors of prescribing were estimated using logistic regression in a matched patient cohort (1:1 by age, sex and calendar time). Results Over 8 million patient records were examined in 587 UK general practices. Practices varied considerably in their propensity to prescribe antibiotics and this variance increased over time. Change points in prescribing did not reflect updates to national guidelines. Prescribing levels within practices were not consistent for different infectious conditions. A history of antibiotic use significantly increased the risk of receiving a subsequent antibiotic (by 22%–48% for patients with three or more antibiotic prescriptions in the past 12 months), as did higher BMI, history of smoking and flu vaccinations. Other drivers for receiving an antibiotic varied considerably for each condition. Conclusions Large variability in antibiotic prescribing between practices and within practices was observed. Prescribing guidelines alone do not positively influence a change in prescribing, suggesting more targeted interventions are required to optimize antibiotic prescribing in the UK.

In the UK, 81% of all antibiotics are prescribed in primary care. Previous research has shown that a letter from the Chief Medical Officer (CMO) giving social norms feedback to General Practitioners (GPs) whose practices are high prescribers of antibiotics can decrease antibiotic prescribing. The aim of this study was to understand the best way for engaging with GPs to deliver feedback on prescribing behaviour that could be replicated at scale, and explore GP information requirements that would be needed to support prescribing behaviour change. Two workshops were devised utilising a participatory approach. Discussion points were noted and agreed with each group of participants. Minutes of the workshops and observation notes were taken. Data were analysed thematically. Four key themes emerged through the data analysis: (1) Our day-to-day reality, (2) GPs are competitive, (3) Face-to-face support, and (4) Empowerment and engagement. Our findings suggest there is potential for using behavioural science in the form of social norms as part of a range of engagement strategies in reducing antibiotic prescribing within primary care. This should include tailored and localised data with peer-to-peer comparisons.

IntroductionThe learning healthcare system (LHS) underpinned by data analysis and feedback to clinical care providers is thought to improve quality of care. The work aimed to implement an LHS for antibiotic prescribing in primary care in England.MethodDeidentified patient-level data from general practices were processed and analysed at regular intervals (fortnightly increments). A dashboard application was developed and implemented displaying analytical graphics to give periodic feedback to clinicians, tailored to each clinical site. Benchmarking parameters were established by the analysis of two large national primary care datasets allowing peer-to-peer comparisons. To date, the dashboard is available to 70 English practices.ConclusionsSuccessful implementation and uptake of the secure technical LHS infrastructure for the analysis and feedback to clinicians of their antibiotic prescribing demonstrate a great appetite for this type of frequent prescribing review in primary care, combining advanced data analytics with tailored feedback.

Background Antimicrobial resistance is driven by the overuse of antibiotics. This study aimed to develop and validate clinical prediction models for the risk of infection-related hospital admission with upper respiratory infection (URTI), lower respiratory infection (LRTI) and urinary tract infection (UTI). These models were used to investigate whether there is an association between the risk of an infection-related complication and the probability of receiving an antibiotic prescription. Methods The study used electronic health record data from general practices contributing to the Clinical Practice Research Datalink (CPRD GOLD) and Welsh Secure Anonymised Information Linkage (SAIL), both linked to hospital records. Patients who visited their general practitioner with an incidental URTI, LRTI or UTI were included and followed for 30 days for hospitalisation due to infection-related complications. Predictors included age, gender, clinical and medication risk factors, ethnicity and socioeconomic status. Cox proportional hazards regression models were used with predicted risks independently validated in SAIL. Results The derivation and validation cohorts included 8.1 and 2.7 million patients in CPRD and SAIL, respectively. A total of 7125 (0.09%) hospital admissions occurred in CPRD and 7685 (0.28%) in SAIL. Important predictors included age and measures of comorbidity. Initial attempts at validating in SAIL (i.e. transporting the models with no adjustment) indicated the need to recalibrate the models for age and underlying incidence of infections; internal bootstrap validation of these updated models yielded C-statistics of 0.63 (LRTI), 0.69 (URTI) and 0.73 (UTI) indicating good calibration. For all three infection types, the rate of antibiotic prescribing was not associated with patients’ risk of infection-related hospital admissions. Conclusion The risk for infection-related hospital admissions varied substantially between patients, but prescribing of antibiotics in primary care was not associated with risk of hospitalisation due to infection-related complications. Our findings highlight the potential role of clinical prediction models to help inform decisions of prescribing of antibiotics in primary care.

Background Previous research reported that individuals prescribed antibiotics frequently develop antimicrobial resistance. The objective of this study was to evaluate whether frequent antibiotic use is associated with reduced hospital admissions for infection-related complications. Methods Population-based cohort study analysing electronic health records from primary care linked to hospital admission records. The study population included patients prescribed a systemic antibiotic, recent record of selected infections and no history of chronic obstructive pulmonary disease. Propensity-matched cohorts were identified based on quintiles of prior antibiotic use in 3 years before. Results A total of 1.8 million patients were included. Repeated antibiotic use was frequent. The highest rates of hospital admissions for infection-related complications were observed shortly after antibiotic start in all prior exposure quintiles. For patients with limited prior antibiotic use, rates then dropped quickly and substantially. In contrast, reductions over time were substantially less in patients with frequent prior antibiotic use, with rates remaining elevated over the following 6 months. In patients without comorbidity comparing the highest to lowest prior exposure quintiles in the Clinical Practice Research Databank, the IRRs were 1.18 [95% CI 0.90–1.55] in the first 3 days after prescription, 1.44 [95% CI 1.14–1.81] in the days 4–30 after and 3.22 [95% CI 2.29–4.53] in the 3–6 months after. Conclusions Repeated courses of antibiotics, although common practice, may have limited benefit and indicator of adverse outcomes. A potential mechanism is that antibiotics may cause dysbiosis (perturbations of intestinal microbiota), contributing to colonization with resistant bacteria. Antibiotics should be used judiciously and only periodically unless indicated. Antimicrobial stewardship should include activities focusing on the substantive number of patients who repeatedly but intermittently get antibiotics.

Objectives To identify the rates of potentially inappropriate antibiotic choice when prescribing for common infections in UK general practices. To examine the predictors of such prescribing and the clustering effects at the practice level. Methods The rates of potentially inappropriate antibiotic choice were estimated using 1 151 105 consultations for sinusitis, otitis media and externa, upper respiratory tract infection (URTI) and lower respiratory tract infection (LRTI) and urinary tract infection (UTI), using the Clinical Practice Research Datalink (CPRD). Multilevel logistic regression was used to identify the predictors of inappropriate prescribing and to quantify the clustering effect at practice level. Results The rates of potentially inappropriate prescriptions were highest for otitis externa (67.3%) and URTI (38.7%) and relatively low for otitis media (3.4%), sinusitis (2.2%), LRTI (1.5%) and UTI in adults (2.3%) and children (0.7%). Amoxicillin was the most commonly prescribed antibiotic for all respiratory tract infections, except URTI. Amoxicillin accounted for 62.3% of prescriptions for otitis externa and 34.5% of prescriptions for URTI, despite not being recommended for these conditions. A small proportion of the variation in the probability of an inappropriate choice was attributed to the clustering effect at practice level (8% for otitis externa and 23% for sinusitis). Patients with comorbidities were more likely to receive a potentially inappropriate antibiotic for URTI, LRTI and UTI in adults. Patients who received any antibiotic in the 12 months before consultation were more likely to receive a potentially inappropriate antibiotic for all conditions except otitis externa. Conclusions Antibiotic prescribing did not always align with prescribing guidelines, especially for URTIs and otitis externa. Future interventions might target optimizing amoxicillin use in primary care.

Background: In mice models of cancer and human tissues samples, disturbance of the gut microbiota can reduce the effectiveness of chemotherapy and immunotherapy. There is limited evidence in humans whether antibiotics impact on the effectiveness of cancer treatments. We evaluated the associations between antibiotic use before cancer diagnoses and long-term mortality. Methods: Primary care data from general practices contributing to the English Clinical Practice Research Datalink (CPRD) and Welsh Secure Anonymised Information Linkage (SAIL) linked to Cancer Registries (CR) and Office for National Statistics (ONS) mortality records were used. Two cohort studies involving cancer patients (15 types) exposed to antibiotics before cancer diagnosis were identified. Recent, previous and past antibiotic users were compared using Cox regression models. Findings: 129 990 cancer patients from the National Cancer Registration and Analysis Service (NCRAS) linked to CPRD and 131 330 from the Welsh Cancer Intelligence Surveillance Unit (WCISU) were included. In comparison with past antibiotic users, patients who used antibiotics in the recent past before their cancer diagnosis and were mainly treated with chemotherapy or immunotherapy presented increased hazards ratio (HR) for mortality consistently in both cohorts; for leukaemia, the HR in NCRAS was 1·40 (95% CI 1·22-1·60), for lymphoma 1·19 (95% CI 1·06-1·33), for melanoma 1·31 (95% CI 1·12-1·54), and for myeloma 1·19 (95% CI 1·04-1·36). HR for cancer of the uterus, breast, urinary, colorectal, and kidney cancer followed the same trend in both CRs. Sensitivity analyses based on general practice records suggested comparable results to those from CRs. Interpretation: This study supports the hypothesis that prior use of antibiotics may reduce the effectiveness of chemotherapy and immunotherapy, potentially through disruption to the gut microbiota. Although not providing definitive results, the biological plausibility of the findings does support the need for judicious use of antibiotics. Funding: Department of Health and NIHR. Declaration of Interest: We declare no competing interests. Ethical Approval: The protocol was approved by the Independent Scientific Advisory Committee for CPRD research (protocol number 18_198) and SAIL’s Information Governance Protocol Review Panel (protocol number 0855).

BackgroundHigh levels of antibiotic prescribing are a major concern as they drive antimicrobial resistance. It is currently unknown whether practices that prescribe higher levels of antibiotics also prescribe more medicines in general.AimTo evaluate the relationship between antibiotic and general prescribing levels in primary care.Design and settingCross-sectional study in 2014–2015 of 6517 general practices in England using NHS digital practice prescribing data (NHS-DPPD) for the main study, and of 587 general practices in the UK using the Clinical Practice Research Datalink for a replication study.MethodLinear regression to assess determinants of antibiotic prescribing.ResultsNHS-DPPD practices prescribed an average of 576.1 antibiotics per 1000 patients per year (329.9 at the 5th percentile and 808.7 at the 95th percentile). The levels of prescribing of antibiotics and other medicines were strongly correlated. Practices with high levels of prescribing of other medicines (a rate of 27 159.8 at the 95th percentile) prescribed 80% more antibiotics than low-prescribing practices (rate of 8815.9 at the 5th percentile). After adjustment, NHS-DPPD practices with high prescribing of other medicines gave 60% more antibiotic prescriptions than low-prescribing practices (corresponding to higher prescribing of 276.3 antibiotics per 1000 patients per year). Prescribing of non-opioid painkillers and benzodiazepines were also strong indicators of the level of antibiotic prescribing. General prescribing levels were a much stronger driver for antibiotic prescribing than other risk factors, such as deprivation.ConclusionThe propensity of GPs to prescribe medications generally is an important driver for antibiotic prescribing. Interventions that aim to optimise antibiotic prescribing will need to target general prescribing behaviours, in addition to specifically targeting antibiotics.

ObjectiveDetermine the association of incident antibiotic prescribing levels for common infections with infection-related complications and hospitalisations by comparing high with low prescribing general practitioner practices.Design retrospective cohort studyRetrospective cohort study.Data sourceUK primary care records from the Clinical Practice Research Datalink (CPRD GOLD) and SAIL Databank (SAIL) linked with Hospital Episode Statistics (HES) data, including 546 CPRD, 346 CPRD-HES and 338 SAIL-HES practices.ExposuresInitial general practice visit for one of six common infections and the proportion of antibiotic prescribing in each practice.Main outcome measuresIncidence of infection-related complications (as recorded in general practice) or infection-related hospital admission within 30 days after consultation for a common infection.ResultsA practice with 10.4% higher antibiotic prescribing (the IQR) was associated with a 5.7% lower rate of infection-related hospital admissions (adjusted analysis, 95% CI 3.3% to 8.0%). The association varied by infection with larger associations in hospital admissions with lower respiratory tract infection (16.1%; 95% CI 12.4% to 19.7%) and urinary tract infection (14.7%; 95% CI 7.6% to 21.1%) and smaller association in hospital admissions for upper respiratory tract infection (6.5%; 95% CI 3.5% to 9.5%) The association of antibiotic prescribing levels and hospital admission was largest in patients aged 18–39 years (8.6%; 95% CI 4.0% to 13.0%) and smallest in the elderly aged 75+ years (0.3%; 95% CI −3.4% to 3.9%).ConclusionsThere is an association between lower levels of practice level antibiotic prescribing and higher infection-related hospital admissions. Indiscriminately reducing antibiotic prescribing may lead to harm. Greater focus is needed to optimise antibiotic use by reducing inappropriate antibiotic prescribing and better targeting antibiotics to patients at high risk of infection-related complications.

Antimicrobial resistance is an important public health concern. As most antibiotics are prescribed in primary care, understanding prescribing patterns in General Medical (GP) practices is vital. The aim of this study was a spatial pattern analysis of antibiotic prescribing rates in GP practices in England and to examine the association of potential clusters with area level socio-economic deprivation. The pattern analysis identified a number of hot and cold spots of antibiotic prescribing, with hot spots predominantly in the North of England. Spatial regression showed that patient catchments of hot spot practices were significantly more deprived than patient catchments of cold spot practices, especially in the domains of income, employment, education and health. This study suggests the presence of area level drivers resulting in clusters of high and low prescribing. Consequently, area level strategies may be needed for antimicrobial stewardship rather than national level strategies.

Fetal growth restriction (FGR) is a serious pregnancy complication, resulting in significant perinatal morbidity and mortality. Increased vascular resistance in the fetoplacental circulation is a hallmark of FGR and is associated with enhanced vasoconstriction of the resistance arteries in the placenta, the chorionic plate arteries (CPAs). Although the cause is unknown, FGR is associated with excess exposure to glucocorticoids (GCs), key mediators of vascular resistance in the systemic circulation. We hypothesized that GCs alter CPA reactivity, thereby contributing to the altered blood flow dynamics seen in FGR. We aimed to examine the acute and chronic effects of GCs on CPA reactivity and the operational mechanisms. Glucocorticoid receptors were highly expressed by CPA. 11β-Hydroxysteroid isoenzyme type 2 was detected within the endothelium, whereas 11β-hydroxysteroid isoenzyme type 1 was absent. Acute GC treatment significantly attenuated U46619-induced constriction. This effect was reversed by cotreatment with mifepristone or an endothelial NOS inhibitor. In contrast, chronic GC treatment potentiated U46619 constriction in a dose-dependent manner, which was partially abolished by mifepristone cotreatment. Similar effects were observed using a novel nonsteroidal glucocorticoid receptor-specific agonist. Chronic treatment with GCs altered the expression of several vasoactive factors, including thromboxane and bradykinin receptors, prokineticin-1, cyclooxygenase-2, and endothelial NOS. In summary, acute and chronic GC treatment exerts contrasting effects on CPA vasoreactivity. These opposing effects are consistent with temporal actions in other vascular beds and reflect activation of distinct nongenomic and genomic pathways. Chronic exposure to elevated GCs may contribute to the raised vascular resistance observed in the fetoplacental circulation in FGR.

s / Placenta 35 (2014) A1eA112 A92 being the catalytic a subunit and the regulatory bII subunit the proteins mainly located in the inner membrane. The experiments with specific inhibitors of cyclases, phosphatases and kinases in JEG-3 cells (choriocarcinoma cells) are in process. 1. Endocrinology, 1997, 138, 2172-83 P2.98-N. THE HEXOSAMINE BIOSYNTHETIC PATHWAY: A ROLE IN NUTRIENT REGULATION OF GROWTH SIGNALLING IN THE HUMAN PLACENTA Victoria Palin, Ian O'Neil, Jim Warwicker, Sterre Paijens, John Aplin, Melissa Westwood The University of Manchester, Manchester, UK Objective: Glucose or glucosamine-stimulated flux through the hexosamine biosynthetic pathway (HBP) leads to the post-translational addition of N-acetyl-glucosamine (GlcNAc) at Ser/Thr residues of intracellular proteins (O-GlcNAcylation), influencing their function by altering location, interactions and phosphorylation. We have previously shown that increased HBP flux inhibits trophoblast proliferation; here we investigate the pathways responsible. Methods: An O-GlcNAcylation site prediction algorithm, developed using information on amino acid sequences flanking published O-GlcNAcylated residues, was used to screen proteins known to influence trophoblast proliferation. BeWo cells were cultured in serum-free DMEM/F12 containing glucose (5-25mM) or glucosamine (0.5-10mM) for 24h, then switched to control medium for a further 48h. Cell number and viability were assessed by counting and trypan-blue exclusion. Global protein GlcNAcylation was confirmed by Western blotting using an anti-O-GlcNAc antibody. The localisation and GlcNAcylation status of tyrosine-protein phosphatase non-receptor type 11 (SHP-2) were assessed by immunocytochemistry and Western blotting using SHP-2 immunoprecipitates with an anti-O-GlcNAc antibody or wheat-germ agglutinin (WGA) respectively. Results: Protein GlcNAcylation increased with increasing glucosamine/ glucose. 24h exposure to 10mM glucosamine led to BeWo cell death; lower concentrations (2.5-5mM) inhibited growth and cells returned to control medium remained in a slower proliferative cycle for at least 48h. 25mM glucose also inhibited proliferation. The prediction algorithm revealed potential O-GlcNAcylation sites in numerous transcription factors and signalling molecules, including Ser/Thr sites in SHP-2 that are close to Tyr residues needed for its activation. Blotting of SHP-2 immunoprecipitates with an anti-O-GlcNAc antibody orWGA demonstrated O-GlcNAcylation of SHP-2. Punctate SHP-2 membrane and cytoplasmic staining was present under all conditions tested suggesting glucose/glucosamine treatment does not affect its expression/localisation. Conclusion: SHP-2, a known regulator of trophoblast proliferation, is posttranslationally modified by O-GlcNAc; regulation of transcription factors / signalling molecules by GlcNAcylation suggests a mechanism by which placental growth can be altered by nutrient availability. P2.99-N. MIR-21 AND ITS ROLE ON TROPHOBLAST CELLS BY TARGETING PHOSPHATASE TENSIN HOMOLOG (PTEN) AND PROGRAMMED CELL DEATH 4 (PDCD4) Wittaya Chaiwangyen, Stephanie Ospina-Prieto, Diana M. MoralesPrieto, Ekkehard Schleussner, Udo R. Markert Placenta-Lab, Department of Obstetrics, University Hospital Jena, Jena, Germany Objectives:MicroRNAs (miRNAs) are a class of small non-coding RNAs (2124 nucleotides) that regulate gene expression by translational inhibition or transcript degradation via targeting the 3’ untranslated region of messenger RNA. MiR-21 is an oncomir and has been associated with various types of cancer. Our group has reported miR-21 as the highest expressed miRNA, out of 762 analyzed, in first trimester isolated trophoblast cells. In this study, we investigated the functions of miR-21 in trophoblastic cell lines and its possible target genes. Methods: The immortalized human trophoblast cell line HTR-8/SVneo and the choriocarcinoma cell line JEG-3 were transfectedwithmiR-21 inhibitor or mimic. Total RNA was extracted and RNA quality and quantity were determined by spectrophotometer. MiRNA-21 expression was quantified by qPCR. Cell growth and invasion were measured after treatment with miR-21 inhibitor or mimic for up to 72 h by using a colorimetric cell proliferation and a Matrigel invasion assay. Migration was assessed by a transwell migration assay and apoptosis by flow cytometry. The expression of the potential targets phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) of miR-21 was analyzed by RT-PCR and Western blotting. Results: Constitutive expression of miR-21 was higher in HTR-8/SVneo cells than in JEG-3 cells. MiR-21 inhibition in JEG-3 or HTR-8/SVneo cells significantly decreased proliferation, while overexpression resulted in a significantly increased proliferation. Invasiveness of JEG-3 cells was significantly reduced after silencing of miR-21. Overexpression of miR-21 significantly inducedmigration in HTR-8/SVneo cells but not in JEG-3 cells. Inhibition of miR-21 expression increased apoptosis in JEG-3 cells, but not in HTR-8/SVneo cells. Silencing of miR-21 enhanced expression of PDCD4 in JEG-3, but not in HTR8/SVneo cells, and PTEN expression in HTR-8/ SVneo, but not in JEG-3 cells. Overexpression of miR-21 significantly induced the opposite effects on the respective proteins in both cell lines. Conclusion: Both cell lines express miR-21 but at different levels. MiR-21 is involved in regulating cell growth, migration, invasion and apoptosis in trophoblast cells potentially by controling the intracellular signalling via PTEN or PDCD4. P2.100. RREPROGRAMMING BY GENE INDUCTION: THE FACTORS INVOLVED IN THE ESTABLISHMENT OF CANINE STEM CELLS Natalia Goncalves, Fabiana Bressan, Fl avio Meirelles, Carlos Ambr osio Faculty of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, Brazil Takahashi and Yamanaka established a technique where transcription factors related to pluripotency were incorporated in the genome of somatic cells in order to reprogram these cells. The expression of these transcription factors enables a somatic cell that presents a differentiated condition to reverse it to an embryonic state, generating induced pluripotent stem cells (iPSC). The production of iPSC from canine fetal fibroblasts was performed with lentiviral integrative systems by policistronic humam and mouse vectors (Stemcca), aiming to obtain cultures of pluripotent stem cells that would develop patterns of gene expression and epigenetic similar to cells derived from the animal model. Canine iPSC lines were produced and these clonal lines were cultivated with bFGF and independently of LIF and presented morphological characteristics that are more close to iPSCs produced in other species such as bovine than to previously described canine iPSC. In the first day after transduction, no differences were observed between murine and human infection. The cells in the reprogramming process presented early colonies at 11 days after transduction, who kept stable and similar to the stem cell morphological patterns. These colonies were picked by hand at the first passage and thereafter Tryple was successfuly used. Such cell lines were alkaline phosphatase positive, had the ability to form embryonic bodies, to spontaneously differentiate and to express pluripotency-related genes (Oct4, Sox2). This work demonstrates it was possible to efficiently reprogram canine somatic cells using lentiviral vectors and proves these cell lines are pluripotent through posive results on the pluripotency tests. Also, these new results combining the use of Tryple enzyme and bFGF supplementation only, unlike what has been done for this specie. P2.101-N. INTERACTIONS OF UMBILICAL MESENCHYMAL STEM CELLS WITH UTERINE AND UMBILICAL ENDOTHELIAL CELLS Neven Ebrahim, Jennifer Sedcole, Olivia Volk, Lopa Leach School of Life Sciences, Nottingham University, Nottingham, UK

Background Fetal growth restriction (FGR) is associated with glucocorticoid (GC) excess in human pregnancies and animal models. In mice and rats, GC treatment reduces placental angiogenesis and dysregulates the expression of angiogenic factors. It is not known whether GCs reduce angiogenesis in the human placenta. Hypothesis Glucocorticoid excess in the human placenta inhibits angiogenesis by dysregulating angiogenic factors. Methods Human umbilical vein endothelial cells (HUVECs) and human placental artery endothelial cells (HPAECs) were treated with hydrocortisone (HC), prednisolone (PRED) and dexamethasone (DEX) for 24–48 hours. Tube-like structure (TLS) formation on matrigel, cell migration, proliferation and apoptosis were assessed. Chorionic plate arteries (CPAs) from normal placentas (n = 10) were cultured for 48 hours with HC or DEX. mRNA expression of six angiogenic factors were quantified using real-time Q-PCR with normalisation to TBP. Results Pilot studies in HUVECs (n = 3, p Discussion GCs reduced tube formation and cell migration, key facets of angiogenesis, in HUVEC and HPAEC models. These findings indicate that GCs inhibit human placental angiogenesis, which could contribute to the pathogenesis of FGR. The downregulation of specific angiogenic factors by GCs identifies putative mechanistic pathways involved.