An investigation into the role of microvesicles in mutant p53 invasive gain-of-function

p53 is a transcription factor with tumour suppressive attributes which is known to be mutated in over half of human cancers. As well as compromising the ability of p53 to function as a transcription factor, mutations in p53 often result in a gain-of-function phenotype which is characterised by increased ability of cancer cells to migrate and invade. This is mediated by the ability of mutant p53 to increase recycling of α5β1 integrin and receptor tyrosine kinases (RTK) from endosomes to the plasma membrane; a process which is dependent on the Rab11 effector, Rab Coupling Protein (RCP) and the phosphatidic acid generating enzyme, diacylglycerol kinase-α (DGKα). Despite accumulating evidence linking RCP/DGKα-dependent receptor recycling to invasive migration, the mechanisms by which mutant p53 controls endosomal trafficking were still unclear when the current study was instigated. Initial experiments indicated that the mutant p53 gain-of-function phenotype was not cell autonomous, and could be passed to p53 null cells by incubating them with conditioned medium from mutant p53 (R273H)-expressing cells. Furthermore, fractionation approaches indicated that the mutant p53 phenotype was transmitted between cells by a microvesicle vector. Upon treatment with microvesicles collected from mutant p53 expressing cells, p53 null cells displayed increased α5β1 integrin and RTK recycling and the consequent invasive/migratory behaviour that was dependent on these RCP and DGKα-regulated trafficking events. Despite a requirement for RCP in the response of p53 null cells to microvesicles, this Rab11 effector was not required for the production of pro-invasive microvesicles. Rather, mutant p53-expressing cells relied on Rab35 (but not Rab27a or Rab27b) for the production and/or release of microvesicles that were capable of transferring mutant p53’s gain-of-function phenotype. An in-depth RNA sequencing analysis indicated that microvesicles from mutant p53 cells influenced the endocytic trafficking and migratory characteristics of p53 null cells without detectably altering mRNA expression in these recipient cells. This indicated the possibility that microvesicles from mutant p53-expressing cells may act directly on the endomembrane system of recipient cells. Immunoprecipitation experiments indicated that there was a physical interaction between Rab35 and podocalyxin (PODXL), a highly-charged sialomucin which is known to directly influence membrane organisation. Additionally, PODXL was detectable in microvesicular preparations by mass spectrometry. Microvesicles purified from mutant p53-expressing cells in which PODXL had been knocked down using siRNA, had significantly reduced capacity to promote integrin/RTK recycling and mutant p53-like migratory behaviour in p53 null cells, indicating that PODXL, as well as Rab35, is a key factor responsible for transmitting mutant p53’s gain-of-function phenotype between cells. In addition to being incapable of influencing the migration of other cells, Rab35 knockdown cells themselves migrated with the characteristics of p53 null cells. Interestingly, microvesicles from mutant p53-expressing cells restored mutant p53-like migratory behaviour in these Rab35 knockdown cells. These data indicate that Rab35 and PODXL-dependent production of phenotype altering microvesicles not only influences the migration of neighbouring cells in a paracrine fashion, but may constitute an autocrine link between mutant p53 and integrin trafficking in the mutant p53 cells themselves. Finally, I have found that p53 null cells may be educated by microvesicles from mutant p53-expressing cells to themselves release cell migration-altering microvesicles, providing further evidence supporting the existence of microvesicle-based autocrine/paracrine mechanisms that may act to propagate mutant p53’s invasive gain-of-function within both homogeneous and heterogeneous populations of tumour cells

The promiscuous public? Exploring public opinion and why it matters to political actors

Political actors often cite public opinion to provide support for public policy decisions. This process is made more challenging with diverse demands and perspectives of the public. How then do political actors decide which opinion gets heard? In this article, we go beyond the assumption that the practice of political representation is indistinguishable across various levels of political actors and ask, why do political actors value public opinion and how does it then influence the way in which they apply this information? Developing a multi-level approach, we employ semi-structured interviews with a wide range of political actors, including politicians, pollsters, and community activists. We find that motivations for defining and applying public opinion differs according to the hierarchy of political actors, demonstrating that the relationship between public and political actors is more nuanced and complex than what is often depicted. In particular, we find that minority views play just an important view in policymaking

Interim estimates of the effectiveness of seasonal trivalent inactivated influenza vaccine in preventing influenza hospitalisations and primary care visits in Auckland, New Zealand, in 2014

We present preliminary results of influenza vaccine effectiveness (VE) in New Zealand using a case test- negative design for 28 April to 31 August 2014. VE adjusted for age and time of admission among all ages against severe acute respiratory illness hospital presentation due to laboratory-confirmed influenza was 54% (95% CI: 19 to 74) and specifically against A(H1N1) pdm09 was 65% (95% CI:33 to 81). For influenza-con- firmed primary care visits, VE was 67% (95% CI: 48 to 79) overall and 73% (95% CI: 50 to 85) against A(H1N1) pdm09

Effectiveness of seasonal trivalent inactivated influenza vaccine in preventing influenza hospitalisations and primary care visits in Auckland, New Zealand, in 2013

This study reports the first vaccine effectiveness (VE) estimates for the prevention of general practice visits and hospitalisations for laboratory-confirmed influenza from an urban population in Auckland, New Zealand, in the same influenza season (2013). A case test-negative design was used to estimate propensity-adjusted VE in both hospital and community settings. Patients with a severe acute respiratory infection (SARI) or influenza-like illness (ILI) were defined as requiring hospitalisation (SARI) or attending a general practice (ILI) with a history of fever or measured temperature ≥38 °C, cough and onset within the past 10 days. Those who tested positive for influenza virus were cases while those who tested negative were controls. Results were analysed to 7 days post symptom onset and adjusted for the propensity to be vaccinated and the timing during the influenza season. Influenza vaccination provided 52% (95%CI: 32 to 66) protection against laboratory-confirmed influenza hospitalisation and 56% (95%CI: 34 to 70) against presenting to general practice with influenza. VE estimates were similar for all typeand subtypes. This study found moderate effectiveness of influenza vaccine against medically attended and hospitalised influenza in New Zealand, a temperate, southern hemisphere country during the 2013 winter season

Prospectus, March 15, 2006

https://spark.parkland.edu/prospectus_2006/1008/thumbnail.jp

Prospectus, May 3, 2006

https://spark.parkland.edu/prospectus_2006/1014/thumbnail.jp

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

The effectiveness of seasonal trivalent inactivated influenza vaccine in preventing laboratory confirmed influenza hospitalisations in Auckland, New Zealand in 2012

Background: Few studies report the effectiveness of trivalent inactivated influenza vaccine (TIV) in preventing hospitalisation for influenza-confirmed respiratory infections. Using a prospective surveillance platform, this study reports the first such estimate from a well-defined ethnically diverse population in New Zealand (NZ). Methods: A case test-negative design was used to estimate propensity adjusted vaccine effectiveness. Patients with a severe acute respiratory infection (SARI), defined as a patient of any age requiring hospitalisation with a history of a fever or a measured temperature ≥38. °C and cough and onset within the past 7 days, admitted to public hospitals in South and Central Auckland were eligible for inclusion in the study. Cases were SARI patients who tested positive for influenza, while non-cases (controls) were SARI patients who tested negative. Results were adjusted for the propensity to be vaccinated and the timing of the influenza season. Results: The propensity and season adjusted vaccine effectiveness (VE) was estimated as 39% (95% CI 16;56). The VE point estimate against influenza A (H1N1) was lower than for influenza B or influenza A (H3N2) but confidence intervals were wide and overlapping. Estimated VE was 59% (95% CI 26;77) in patients aged 45-64 years but only 8% (-78;53) in those aged 65 years and above. Conclusion: Prospective surveillance for SARI has been successfully established in NZ. This study for the first year, the 2012 influenza season, has shown low to moderate protection by TIV against influenza positive hospitalisation