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The Nuffield Department of Surgical Sciences is the academic department of surgery at the University of Oxford, and hosts a multidisciplinary team of senior clinical academic surgeons, senior scientists, junior clinicians and scientists in training.
The Oncogene Metadherin Interacts with the Known Splicing Proteins YTHDC1, Sam68 and T-STAR and Plays a Novel Role in Alternative mRNA Splicing.
Oncogenic metadherin is a key contributor to tumourigenesis with metadherin expression and cytoplasmic localisation previously linked to poor survival. A number of reports have shown metadherin localises specifically to nuclear speckles known to be rich in RNA-binding proteins including the splicing proteins YTHDC1, Sam68 and T-STAR, that have been shown to select alternative splice sites in mRNA of tumour-associated proteins including BRCA, MDM2 and VEGF. Here we investigate the interaction and relationship between metadherin and the splice factors YTHDC1, T-STAR and Sam68. Using a yeast two-hybrid assay and immunoprecipitation we show that metadherin interacts with YTHDC1, Sam68 and T-STAR and demonstrate that T-STAR is significantly overexpressed in prostate cancer tissue compared to benign prostate tissue. We also demonstrate that metadherin influences splice site selection in a dose-dependent manner in CD44v5-luc minigene reporter assays. Finally, we demonstrate that prostate cancer patients with higher metadherin expression have greater expression of the CD44v5 exon. CD44v5 expression could be used to discriminate patients with poor outcomes following radical prostatectomy. In this work we show for the first time that metadherin interacts with, and modulates, the function of key components of splicing associated with cancer development and progression.
Drivers of AR indifferent anti-androgen resistance in prostate cancer cells.
Inhibition of the androgen receptor (AR) by second-generation anti-androgens is a standard treatment for metastatic castration resistant prostate cancer (mCRPC), but it inevitably leads to the development of resistance. Since the introduction of highly efficient AR signalling inhibitors, approximately 20% of mCRPC patients develop disease with AR independent resistance mechanisms. In this study, we generated two anti-androgen and castration resistant prostate cancer cell models that do not rely on AR activity for growth despite robust AR expression (AR indifferent). They are thus resistant against all modern AR signalling inhibitors. Both cell lines display cross-resistance against the chemotherapeutic drug docetaxel due to MCL1 upregulation but remain sensitive to the PARP inhibitor olaparib and the pan-BCL inhibitor obatoclax. RNA-seq analysis of the anti-androgen resistant cell lines identified hyper-activation of the E2F cell-cycle master regulator as driver of AR indifferent growth, which was caused by deregulation of cyclin D/E, E2F1, RB1, and increased Myc activity. Importantly, mCRPC tissue samples with low AR activity displayed the same alterations and increased E2F activity. In conclusion, we describe two cellular models that faithfully mimic the acquisition of a treatment induced AR independent phenotype that is cross-resistant against chemotherapy and driven by E2F hyper-activation.
Human-Based Exposure Levels of Perfluoroalkyl Acids May Induce Harmful Effects to Health by Disrupting Major Components Androgen Receptor Signalling In Vitro
© 2019, The Author(s). Perfluoroalkyl acids (PFAAs) are detectable in human blood. PFAA exposure may contribute to androgen receptor (AR)-related health effects such as prostate cancer (PCa). In Norway and Sweden, exposures to PFAAs and PCa are very real concerns. In vitro studies conventionally do not investigate PFAA-induced AR disruption at human blood-based concentrations, thus limiting application to human health. We aim to determine the endocrine disrupting activity of PFAAs based upon human exposure levels, on AR transactivation and translocation. PFAAs (PFOS, PFOA, PFNA, PFDA, PFHxS, and PFUnDA) were tested at concentrations ranging from 1/10 × to 500 × relative to human blood based upon the exposure levels observed in a Scandinavian population. Translocation was measured by high content analysis (HCA) and transactivation was measured by reporter gene assay (RGA). No agonist activity (translocation or transactivation) was detected for any PFAAs. In the presence of testosterone, AR translocation increased following exposure to PFOS 1/10 × and 100 ×, PFOA 1/10 ×, and PFNA 1 × and 500 × (P < 0.05). In the presence of testosterone, PFOS 500 × antagonised AR transactivation, whereas PFDA 500 × increased AR transactivation (P < 0.05). PFAAs may contribute to AR-related adverse health effects such as PCa. PFAAs can disrupt AR signalling via two major components: translocation and transactivation. PFAAs which disrupt one signalling component do not necessarily disrupt both. Therefore, to fully investigate the disruptive effect of human exposure-based contaminants on AR signalling, it is imperative to analyse multiple molecular components as not all compounds induce a disruptive effect at the same level of receptor signalling.
Surveillance arterioveNous fistulAs using ultRasound (SONAR) trial in haemodialysis patients: a study protocol for a multicentre observational study.
INTRODUCTION: Arteriovenous fistulas (AVFs) are considered the best and safest modality for providing haemodialysis in patients with end-stage renal disease. Only 20% of UK centres achieve the recommended 80% target for achieving dialysis of the prevalent dialysis population via permanent access (as opposed to a central venous catheter). This is partly due to the relatively poor maturation rate of newly created fistulas, with as many as 50% of fistulas failing to mature.The Surveillance Of arterioveNous fistulAe using ultRasound study will examine whether a protocolised programme of Doppler ultrasound (US) surveillance can identify, early after creation, potentially correctable problems in those AVFs that subsequently fail to mature. METHODS AND ANALYSIS: This is a multicentre observational study that will assess newly created AVFs by Doppler US performed at 2, 4, 6 and 10 weeks after creation. The primary outcome measure will be primary fistula patency at week 10. Secondary outcome measures include: successful use of the fistula; clinical suitability for dialysis; creation of new fistula or radiological salvage; fistula thrombosis; secondary fistula patency rate and patient acceptability. ETHICS AND DISSEMINATION: The study has been approved by the Cambridgeshire and Hertfordshire Research Ethics Committee and by the Health Research Authority (REC 18/EE/0234). The results generated from this work will be published as open access, within 3 years of trial commencement. We will also present our findings at key national/international renal meetings, as well as support volunteers at renal patient groups to disseminate the trial outcome. TRIAL REGISTRATION NUMBER: ISRCTN36033877.
CD8+ Immunosenescence Predicts Post-Transplant Cutaneous Squamous Cell Carcinoma in High-Risk Patients.
Most morbidity associated with malignancy in long-term renal transplant recipients is due to cutaneous squamous cell carcinoma (SCC). Previously identified measures to stratify SCC risk have limited use, however. We hypothesized that an increased proportion of senescent, terminally differentiated CD8(+) T cells would identify renal transplant recipients at elevated SCC risk. Peripheral blood lymphocytes were isolated from 117 stable transplant recipients at high risk of SCC and analyzed phenotypically by flow cytometry. Participants were followed up prospectively for SCC development. The predictive value of variables was assessed using Cox regression. Age at transplant and enrollment, dialysis duration, and previous disease were predictive of SCC development during follow-up. Previously published clinical phenotype-based risk scores lost predictive value with the removal of age as a covariate. The percentage of CD57-expressing CD8(+) T cells was the strongest immunologic predictor of future SCC and correlated with increasing CD8(+) T cell differentiation. We dichotomized participants into those with a majority (CD57hi) and a minority (CD57lo) of CD8(+) T cells expressing CD57; CD57hi participants were more likely to develop SCC during follow-up (hazard ratio, 2.9; 95% confidence interval, 1.0 to 8.0), independent of potential confounders, and tended to develop earlier recurrence. The CD57hi phenotype was stable with time and associated with increasing age and cytomegalovirus seropositivity. Our results show that the CD57hi phenotype is a strong predictor of SCC development and recurrence in this cohort of long-term, high-risk renal transplant recipients. This information may allow identification of recipients who may benefit from intensive dermatologic screening and immunosuppression reduction.
Application of Operational Tolerance Signatures Are Limited by Variability and Type of Immunosuppression in Renal Transplant Recipients: A Cross-Sectional Study.
BACKGROUND: Renal transplant recipients (RTR) frequently develop complications relating to chronic immunosuppression. Identifying RTR who could safely reduce immunosuppression is therefore highly desirable. We hypothesized that "signatures" described in RTR who have stopped immunosuppression but maintained stable graft function ("operational tolerance") may enable identification of immunosuppressed RTR who are candidates for immunosuppression minimization. However, the effect of immunosuppression itself on these signatures and circulating B-cell populations is currently unknown. METHODS: We undertook a cross-sectional study of 117 RTR to assess the effect of immunosuppression upon circulating B cell populations, humoral alloresponse and 2 previously published "signatures" of operational tolerance. RESULTS: Immunosuppression associated with alterations in both published "signatures." Azathioprine associated with a decrease in transitional and naive B-cell numbers and calcineurin inhibition associated with an increase in the number of circulating plasmablasts. However, only azathioprine use associated with the presence of donor-specific anti-HLA IgG antibodies. Calcineurin inhibition associated with an increase in total serum IgM but not IgG. Data were corrected for age, time since last transplant, and other immunosuppression. CONCLUSIONS: Current signatures of operational tolerance may be significantly affected by immunosuppressive regimen, which may hinder use in their current form in clinical practice. Calcineurin inhibition may prevent the development of long-lasting humoral alloresponses, whereas azathioprine therapy may be associated with donor specific antibody development.
Interruptions during drug rounds: an observational study.
Drug administration errors are a common problem; they contribute to unintended patient harm and may in part be due to distractions during the drug administration round. In this study, a prospective sample of 38 drug rounds on an acute surgical ward were observed, and their duration, time spent dealing with interruptions and nature of the interruptions were recorded. On average, 11% of each drug round was spent dealing with interruptions. There were one or more interruptions in two-thirds of the rounds studied (average 2.61 interruptions per round), with a mean duration of 1 minute per interruption. The interruptions came from doctors (21%), other nurses (17%), patients (11%), telephone enquiries (8%), relatives (3%) or were initiated by the nurse conducting the round (21%). This pattern contradicts the subjective impressions of nurses in previous questionnaire studies, but it was notable that the longest individual interruptions were from conversations with patients (mean 249 seconds) and phone calls (mean 212 seconds). The data confirm the frequency of interruptions, and their potential as a safety hazard. Objective information from direct observation will prove valuable in designing possible solutions to the problem. These will require local knowledge and frontline staff involvement to be sustainable.
Single estimated glomerular filtration rate and albuminuria measurement substantially overestimates prevalence of chronic kidney disease.
BACKGROUND/AIMS: Guidelines require repeatedly diminished estimated glomerular filtration rate (eGFR) and/or albuminuria to diagnose chronic kidney disease (CKD), and advise screening only in select populations. Many estimates of CKD prevalence have used single measurements. This longitudinal study assessed eGFR and albuminuria reproducibility, and impact on estimate of CKD prevalence, in factory workers. METHODS: A total of 512 white workers in a Belarusian industrial factory were initially tested, identifying 206 with abnormal (eGFR <59 ml/min/1.73 m(2) or albuminuria) or near-abnormal (eGFR up to 1 SD above abnormal) renal function. At 3 months, 142 of the abnormal/near-abnormal cohort were re-tested. RESULTS: Analysis of repeat samples revealed no significant change in eGFR in this population, however 21% individually changed CKD stage. Initial proteinuria was reproducible in only 48% at 3 months. This had a major impact on estimated CKD prevalence: a point prevalence of 8.2% halved with repeat testing. The predictive value of initially abnormal eGFR or albuminuria for repeat abnormality at 3 months was 0.5. CONCLUSION: Non-targeted screening for CKD is inaccurate and can overestimate prevalence. This study emphasises the importance of confirming abnormal eGFR and proteinuria on at least one further sample 3 months apart before categorising the individual as having CKD. This has wide implications for screening in European general populations.
Repeat testing is essential when estimating chronic kidney disease prevalence and associated cardiovascular risk.
BACKGROUND: Investigations into chronic kidney disease (CKD) and cardiovascular disease in the CKD population may be misleading as they are often based on a single test of kidney function. AIM: To determine whether repeat testing at 3 months to confirm a diagnosis of CKD impacts on the estimated prevalence of CKD and the estimated 10-year general cardiovascular risk of the CKD population. DESIGN AND METHODS: Blood and urine samples from presumed healthy volunteers were analysed for evidence of CKD on recruitment and again 3 months later. Estimated 10-year cardiovascular risk was calculated using criteria determined by the Framingham study. Preliminary study: 512 volunteers were screened for CKD. Of the initial results, 206 indicated CKD or eGFR within one standard deviation of abnormal, and 142 (69%) of these were retested. Validation study: 528 volunteers were recruited and invited to return for repeat testing. A total of 214 (40.5%) participants provided repeat samples. RESULTS: A single test indicating CKD had a positive predictive value of 0.5 (preliminary) and 0.39 (validation) for repeat abnormalities 3 months later. Participants with CKD confirmed on repeat testing had a significant increase in estimated 10-year cardiovascular risk over the population as a whole (preliminary: 16.5 vs. 11.9%, P < 0.05; validation: 18.1 vs. 9.2%, P < 0.01). Participants with a solitary test indicating CKD had no elevation in cardiovascular risk. CONCLUSION: Repeat testing for CKD after 3 months significantly reduces the estimated prevalence of disease and identifies a population with true CKD and a cardiovascular risk significantly in excess of the general population.
A multi-centre retrospective cohort study comparing the efficacy, safety and cost-effectiveness of hysterectomy and uterine artery embolisation for the treatment of symptomatic uterine fibroids. The HOPEFUL study.
OBJECTIVES: To examine and compare the medium-term results of hysterectomy and uterine artery embolisation (UAE) as a treatment for symptomatic uterine fibroids with regard to safety, efficacy, special issues in the UAE group, cost-effectiveness, and women's own perspectives on the treatments. DESIGN: Data were collected locally from patients' hospital records and also from patients themselves by postal questionnaire. Questionnaire data included free-text comments and this qualitative material was analysed using constant comparison. A two-stage probabilistic decision model was designed to estimate UK NHS costs and health outcomes in terms of quality-adjusted life-years (QALYs). SETTING: Eighteen NHS hospital trusts, 17 in England and one in Scotland. PARTICIPANTS: Eligible women (972 UAE, 762 hysterectomies) who had received treatment specifically for symptomatic fibroids were identified. INTERVENTIONS: The UAE patients were treated by experienced interventional radiologists and all received their index UAE prior to the end of 2002, ensuring a minimum 2-year follow-up. The average length of follow-up was 8.6 years for the hysterectomy cohort and 4.6 years for the UAE cohort. MAIN OUTCOME MEASURES: Primary outcome measures were complication rates to assess the comparative safety of the two interventions. Secondary outcome measures related to treatment efficacy including resolution of symptoms and patient-reported satisfaction with treatment. Further efficacy outcome measures obtained in the UAE group included fibroid/uterine size shrinkage and further treatments required for unresolved fibroid symptoms. Data were also gathered on pregnancies post-UAE. RESULTS: Data were available for 1108 women (649 UAE and 459 hysterectomy). Fewer complications were experienced by women in the UAE cohort compared to the hysterectomy cohort: hysterectomy n = 120 (26.1%), UAE n = 114 (17.6%), adjusted odds ratio 0.48 [95% confidence interval (CI) 0.26 to 0.89]. When only the severe/major complications were considered, this odds ratio was reduced to 0.25 (95% CI 0.13 to 0.48). Expected general side-effects of UAE occurred in 32.7% of the UAE cohort, of which 8.9% also experienced complications. Obesity and medical co-morbidity predisposed women to complications, whereas prophylactic antibiotics appeared to protect against both complications and the expected side-effects of UAE. More women in the hysterectomy cohort reported relief from fibroid symptoms (89% versus 80% UAE, p less than 0.0001) and feeling better (81% versus 74% UAE, p less than 0.0001), but only 70% (compared with 86% UAE, p = 0.007) would recommend their treatment to a friend. In the UAE cohort, 18.3% of the women went on to receive one or more further fibroid treatments including hysterectomy (11.2%). After adjusting for differential time of follow-up, the UAE women had up to a 23% (95% CI 19 to 27%) likelihood of requiring further treatment. The free-text data indicated that many women, in both cohorts, felt that their treatment had been a complete success. In the UAE cohort there were several areas where expectations were apparently high and outcome had not fulfilled their expectations. Disappointment was expressed mainly about continuation or return of symptoms or failure to become pregnant. Many continued to have remaining questions about their treatment. The economic analysis indicated that UAE is less expensive than hysterectomy even after further treatments for unresolved or recurrent symptoms are taken into account, with little difference in QALYs between the two treatments. Younger women are exposed to the risk of recurrent fibroids and subsequent additional procedures over a longer period and consequently UAE may no longer be cost-effective. CONCLUSIONS: The study results suggest that both UAE and hysterectomy are safe. No unexpected problems were detected following UAE after a long follow-up period (average 5 years). Complications are less common for UAE than hysterectomy. The cost-effectiveness analysis favours embolisation even after taking account of complications, expected side-effects associated with the procedure and subsequent re-treatments for women with a preference for uterus preservation. It is important to improve the management of expectations following UAE, particularly regarding fertility. The data suggested that fertility and miscarriage rate are consistent with those of age-matched women with fibroids. UAE is an effective treatment for some women with fibroids and our trial supports the National Institute for Health and Clinical Excellence guidance that it should be made available as one of the options for treatment, with a possible reduction in the need for hysterectomy as the first-line treatment. Further research is needed into which women will be treated most successfully by UAE, the best method of achieving effective embolisation, advice for women who desire future fertility, the role of prophylactic antibiotics in UAE, and the effects of HRT use after UAE on recurrence of fibroid symptoms.
Altered Phenotype of β-Cells and Other Pancreatic Cell Lineages in Patients With Diffuse Congenital Hyperinsulinism in Infancy Caused by Mutations in the ATP-Sensitive K-Channel.
Diffuse congenital hyperinsulinism in infancy (CHI-D) arises from mutations inactivating the KATP channel; however, the phenotype is difficult to explain from electrophysiology alone. Here we studied wider abnormalities in the β-cell and other pancreatic lineages. Islets were disorganized in CHI-D compared with controls. PAX4 and ARX expression was decreased. A tendency toward increased NKX2.2 expression was consistent with its detection in two-thirds of CHI-D δ-cell nuclei, similar to the fetal pancreas, and implied immature δ-cell function. CHI-D δ-cells also comprised 10% of cells displaying nucleomegaly. In CHI-D, increased proliferation was most elevated in duct (5- to 11-fold) and acinar (7- to 47-fold) lineages. Increased β-cell proliferation observed in some cases was offset by an increase in apoptosis; this is in keeping with no difference in INSULIN expression or surface area stained for insulin between CHI-D and control pancreas. However, nuclear localization of CDK6 and P27 was markedly enhanced in CHI-D β-cells compared with cytoplasmic localization in control cells. These combined data support normal β-cell mass in CHI-D, but with G1/S molecules positioned in favor of cell cycle progression. New molecular abnormalities in δ-cells and marked proliferative increases in other pancreatic lineages indicate CHI-D is not solely a β-cell disorder.
Human blood-based exposure levels of persistent organic pollutant (POP) mixtures antagonise androgen receptor transactivation and translocation.
INTRODUCTION: Human exposure to persistent organic pollutants (POPs) has been linked to genitourinary health-related conditions such as decreased sperm quality, hypospadias, and prostate cancer (PCa). Conventional risk assessment of POPs focuses on individual compounds. However, in real life, individuals are exposed to many compounds simultaneously. This might lead to combinatorial effects whereby the global effect of the mixture is different from the effect of the single elements or subgroups. POP mixtures may act as endocrine disruptors via the androgen receptor (AR) and potentially contribute to PCa development. AIM: To determine the endocrine disrupting activity of a POP mixture and sub-mixtures based upon exposure levels detected in a human Scandinavian population, on AR transactivation and translocation in vitro. MATERIALS AND METHODS: The Total POP mixture combined 29 chemicals modelled on the exposure profile of a Scandinavian population and 6 sub-mixtures: brominated (Br), chlorinated (Cl), Cl + Br, perfluorinated (PFAA), PFAA + Br, PFAA + Cl, ranging from 1/10× to 500× relative to what is found in human blood. Transactivation was measured by reporter gene assay (RGA) and translocation activity was measured by high content analysis (HCA), each using stably transfected AR model cell lines. RESULTS: No agonist activity in terms of transactivation and translocation was detected for any POP mixtures. In the presence of testosterone the Cl + Br mixture at 100× and 500× blood level antagonised AR transactivation, whereas the PFAA mixture at blood level increased AR transactivation (P < 0.05). In the presence of testosterone the Cl and PFAA + Br mixtures at 1/10×, 1×, and 50× blood level antagonised AR translocation (P < 0.05). CONCLUSION: Taken together, some combinations of POP mixtures can interfere with AR translocation. However, in the transactivation assay, these combinations did not affect gene transactivation. Other POP combinations were identified here as modulators of AR-induced gene transactivation without affecting AR translocation. Thus, to fully evaluate the effect of environmental toxins on AR signalling, both types of assays need to be applied.