Search

Hypoxia and a suppressive tumour microenvironment (TME) are both independent negative prognostic factors for muscle-invasive bladder cancer (MIBC) that contribute to treatment resistance. Hypoxia has been shown to induce an immune suppressive TME by recruiting myeloid cells that inhibit anti-tumour T cell responses. Recent transcriptomic analyses show hypoxia increases suppressive and anti-tumour immune signalling and infiltrates in bladder cancer. This study sought to investigate the relationship between hypoxia-inducible factor (HIF)-1 and -2, hypoxia, and immune signalling and infiltrates in MIBC. ChIP-seq was performed to identify HIF1α, HIF2α, and HIF1β binding in the genome of the MIBC cell line T24 cultured in 1% and 0.1% oxygen for 24 h. Microarray data from four MIBC cell lines (T24, J82, UMUC3, and HT1376) cultured under 1%, 0.2%, and 0.1% oxygen for 24 h were used. Differences in the immune contexture between high- and low-hypoxia tumours were investigated using in silico analyses of two bladder cancer cohorts (BCON and TCGA) filtered to only include MIBC cases. GO and GSEA were used with the R packages “limma” and “fgsea”. Immune deconvolution was performed using ImSig and TIMER algorithms. RStudio was used for all analyses. Under hypoxia, HIF1α and HIF2α bound to ~11.5–13.5% and ~4.5–7.5% of immune-related genes, respectively (1–0.1% O2). HIF1α and HIF2α both bound to genes associated with T cell activation and differentiation signalling pathways. HIF1α and HIF2α had distinct roles in immune-related signalling. HIF1 was associated with interferon production specifically, whilst HIF2 was associated with generic cytokine signalling as well as humoral and toll-like receptor immune responses. Neutrophil and myeloid cell signalling was enriched under hypoxia, alongside hallmark pathways associated with Tregs and macrophages. High-hypoxia MIBC tumours had increased expression of both suppressive and anti-tumour immune gene signatures and were associated with increased immune infiltrates. Overall, hypoxia is associated with increased inflammation for both suppressive and anti-tumour-related immune signalling and immune infiltrates, as seen in vitro and in situ using MIBC patient tumours.

Background: As hypoxia can drive an immunosuppressive tumour microenvironment and inhibit CD8+ T cells, we investigated if patients with low tumour CD8+ T cells benefitted from hypoxia-modifying therapy. Methods: BCON was a phase III trial that randomised patients with muscle-invasive bladder cancer (MIBC) to radiotherapy alone or with hypoxia-modifying carbogen plus nicotinamide (CON). Tissue microarrays of diagnostic biopsies from 116 BCON patients were stained using multiplex immunohistochemistry (IHC) with the markers CD8, CD4, FOXP3, CD68 and PD-L1, plus DAPI. Hypoxia was assessed using CA9 IHC (n = 111). Linked transcriptomic data (n = 80) identified molecular subtype. Relationships with overall survival (OS) were investigated using Cox proportional hazard models. Results: High (upper quartile) vs. low CD8 T cell counts associated with a better OS across the whole cohort at 16 years (n = 116; HR 0.47, 95% CI 0.28–0.78, p = 0.003) and also in the radiotherapy alone group (n = 61; HR 0.39, 95% CI 0.19–0.76, p = 0.005). Patients with low CD8+ T cells benefited from CON (n = 87; HR 0.63, 95% CI 0.4–1.0, p = 0.05), but those with high CD8 T cells did not (n = 27; p = 0.95). CA9 positive tumours had fewer CD8+ T cells (p = 0.03). Prognostic significance of low CD8+ T cells in the whole cohort remained after adjusting for clinicopathologic variables. Basal vs. luminal subtype had more CD8+ cells (p = 0.02) but was not prognostic (n = 80; p = 0.26). Exploratory analyses with other immune markers did not improve on findings obtained with CD8 counts. Conclusions: MIBC with low CD8+ T cell counts may benefit from hypoxia-modifying treatment.

Introduction Recent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease. Materials and methods Three human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA. Results When T24 cells were seeded at p = 0.009) and mRNA (p p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p p Conclusion Tumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.

Introduction: Hypoxia and an immunosuppressive tumor microenvironment (TME) are both independent negative prognostic factors that contribute to radiotherapy resistance in muscle-invasive bladder cancer (MIBC). Therefore, we sought to investigate the relationship between them and whether the immune TME can predict benefit from hypoxia-modifying therapy in the BCON trial that randomized MIBC patients to radiotherapy alone or with carbogen plus nicotinamide (CON). Methods: Tissue microarrays of diagnostic biopsies from 116 MIBC patients from the BCON trial were stained using Akoya Opal࣪ multiplex immunohistochemistry (IHC) reagents (CD8, CD4, FOXP3, CD68, PDL1, DAPI). Following multispectral scanning the slides were spectrally unmixed using Inform and marker proportion quantified using HALO software as the percentage of the total cell count per sample. Hypoxia was assessed as the presence of CA9 protein using IHC (n=111) and by using transcriptomic data (n=80) previously acquired to determine an hypoxia score (HS). Relationships with overall (OS) and local progression-free (LPFS) survival were assessed using Cox proportional hazard models and Kaplan-Meier curves in R. Molecular subtypes were determined using the consensusMIBC package in R. Results: Basal/squamous and stroma-rich vs other molecular subtypes had increased hypoxia scores and immune markers (CD8, CD4, CD68, PDL1). Tumors with CA9 present had significantly lower proportion of CD8 T cells (p=0.027), and CD4 T cells (p=0.01) but there were no significant differences for other immune markers or significant differences associated with HS. Only CD8 showed a relationship with survival and we found the relationship between log(HR) and CD8 to be non-linear, which led to placing patients within groups. Patients with a low vs high proportion of CD8 T cells had a significantly worse 2-year OS (n=116, p=0.003) and LPFS (n=116, p=0.02). CD8 proportion was also prognostic in patients receiving radiotherapy alone for OS (n=61; p=0.003) and LPFS (n=61; p=0.003). Patients with low CD8 T cells had improved OS when CON was given with radiotherapy (n=87; p=0.05), but those with high CD8 T cells derived no benefit (n=27; p=0.95). Prognostic significance of low CD8 T cells in the whole cohort remained after adjusting for clinicopathologic variables and hypoxia measured using CA9 (HR 0.32; 95% CI 0.18-0.57; p Conclusions: Low proportion of CD8 T cells in the TME of MIBC patients has independent prognostic and predictive value. It can be used to identify patients likely to benefit from the addition of carbogen and nicotinamide to radiotherapy. Citation Format: Vicky Smith, Debayan Mukherjee, Anna Maria Tsakiroglou, Alexander Baker, Hitesh Mistry, Ananya Choudhury, Peter Hoskin, Tim Illidge, Catharine West. Low CD8 T cells predicts benefit from hypoxia-modifying therapy in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6236.

BackgroundAdjuvant use of laser with systemic chemotherapy for treatment of retinoblastoma may reduce recurrence rates while also causing local side effects. Information is lacking on the effect of laser on visual outcomes.MethodsA retrospective review of two retinoblastoma centres in the United Kingdom was conducted. Patients were included if there was a macular tumour in at least one eye. Eyes that received chemotherapy alone were compared with eyes that received chemotherapy plus adjuvant laser.ResultsA total of 76 patients and 91 eyes were included in the study. Systemic chemotherapy alone was used in 71 eyes while chemotherapy plus laser was used in 20 eyes. Demographic characteristics of both groups were similar. Macular relapse rates were similar between groups: 22/71 (31%) eyes in chemotherapy group and 9/20 (45%) eyes in laser group (p=0.29). There was no increase in vitreous relapses in the laser group (2/20 eyes), compared with the chemotherapy group 10/71 eyes (p=0.99). Survival analysis demonstrated similar time to first relapse between groups. Final visual acuity was equal between groups with 6/15 or better present in 31.1% of eyes in the chemotherapy group and 37.5% of eyes in the laser group (p=0.76). Presence of tumour at the fovea was predictive of final visual acuity, regardless of treatment group.ConclusionAdjuvant laser in the treatment of retinoblastoma is safe and does not lead to increased rate of vitreous recurrence. Final visual acuity is determined by the presence of tumour at the fovea and not the use of laser.

Our problem, what we did and how: ESBT is a partnership between two local Clinical Commissioning Groups CCGs, the acute and community NHS Trust, mental health NHS Foundation Trust and the County Council. The partners’ aim is to integrate the whole system to ensure they use their combined £860m annual budget to achieve the best possible services for their local population approximately 370,000 people. To facilitate this in 2017/18 ESBT came up with a transitional governance model within the current UK legislative context, in order to enable the partners work differently together as a whole health and care system to achieve their ESBT objectives. The integration transformation programme of work started in 2013 and the new governing arrangements have been in place since April 2017.Highlights innovation, Impact and outcomes:Tangible outputs of this work to date include for example:A single integrated point of contact hub for all health and care services;A Crisis Response service supporting older people at risk of emergency admission to hospital;Fully staffed and operational integrated locality teams in 6 localities;A welfare benefits Project that has supported over 7,000 people;183schools with developed whole school health improvement plans;136 nurseries – 82% total / rising to 93% in areas with highest inequalities have signed up to the nursery transformation programme Healthy Active Little Ones HALO; rocess and outcome measures that indicate the impact that the programme has had to date include:Activity change between 2013/14 and 2016/17 is lower for ESBT and the acute provider, than in STP CCGs and nationally in all points of delivery bar A&E attendances, however A&E attendances have been reduced in the over 65s;Comparators showing CCG activity rates per 100,000 weighted population against the average for England are lower across almost all points of delivery other than bed days;72% of people seen by the Crisis Response have not been re-admitted to hospital within a month;Improved access to psychological therapies– target 75% actual; Eastbourne Hailsham and Seaford 85.5% / Hastings and Rother 89%Healthy Living PharmacistHLP - 53 pharmacists and 10 pharmacy staff in wider roles attended leadership training in preparation for new ways of working.Conclusions: In order to develop and shape proposals for further strengthening the ESBT Alliance in 2018/19 we have mapped and reviewed the existing governance arrangements of our sovereign commissioning member organisations, as well as the key elements of our new ESBT Alliance governance structure, to further develop our understanding of the following:what is currently delegable to our Alliance and what isn’t;where lay, clinical and elected members’ roles could be used to best effect in assuring plans as we move forward on a system-wide basis in 2018/19;an initial screening exercise of potential equalities and health inequalities impacts, and;the transitional timetable that will be necessary to test system-wide governance and leadership arrangements over the year.The process of developing the new governance model should be transferable to any context.

Drawing on both experimental film theory and eco-philosophy, this thesis finds that much contemporary experimental time-lapse and roto-scope animation stimulates unusual perspectives on objects, manifesting their energies and vibrancies in ways that would otherwise remain imperceptible to the human senses, and engaging therefore with concerns core to current ecological scholarship. While time-lapse sequences in dominant cinema anthropomorphize matter, dissipating its power into ‘secrets’, for example, this chapter suggests that experimental animation—including Proximity (2006) and Talking to a Stone (1993) by Inger Lise Hansen and Gasometers by Nicky Hamlyn (2015)—in considering matter’s own force and vitality, avoids the anthropocentrism often found in nature documentary or character animation.

This book discusses developments and continuities in experimental animation that, since Robert Russet and Cecile Starr’s Experimental Animation: Origins of a New Art (1976), has proliferated in the context of expanded cinema, performance and live ‘making’ and is today exhibited in galleries, public sites and online. With reference to historical, critical, phenomenological and inter-disciplinary approaches, international researchers offer new and diverse methodologies for thinking through these myriad animation practices. This volume addresses fundamental questions of form, such as drawing and the line, but also broadens out to encompass topics such as the inter-medial, post-humanism, the real, fakeness and fabrication, causation, new forms of synthetic space, ecology, critical re-workings of cartoons, and process as narrative. This book will appeal to cross and inter-disciplinary researchers, animation practitioners, scholars, teachers and students from Fine Art, Film and Media Studies, Philosophy and Aesthetics.

Building on phenomenological studies of the body and feminist structural materialist film theory and practice, this article proposes that performed methods of direct-on-film animation shape novel choreographies and produce original filmic patterns and rhythms. The author draws a parallel between Foucault’s disobedient ‘non-normative’ body and non-standardized interventions with film technology. The article analyses the innovative choreographies that arise from rotoscoping techniques, direct-on-film practices and expanded cinema performances. Focusing specifically on Free Radicals (Len Lye, 1958) and Reel Time (Annabel Nicolson, 1973), the author uses these analyses to shed light on her own performance of making films by moving in unusual ways, in time with technologies, and marking directly onto the film surface with the animated body. While engaging with structural materialist questions of film form, process and materials, this article departs from such modernist concerns to propose that the performance of filmmaking offers opportunities to explore the tenacity and vulnerability of particular bodies. The author suggests that performed filmmaking is equally about the traces of actions made on film and the unusual choreography that brings these marks into being.

An article on the London Film-makers’ Co-op second generation Initiated and edited by Michael Maziere and Nina Danino based on a roundtable with Nina Danino, James Mackay, Michael Maziere, Vicky Smith and Chaired by William Fowler.

The Manchester Attachment Story Task (MCAST) is a new method for eliciting the internal working model (IWM) of attachment representations in children between 5 and 7 years. The instrument is described in the companion paper, 'A New Method of Evaluating Attachment Representations'. This paper reports initial findings on a non-clinical sample comparing the MCAST with concurrent maternal attachment representation, measures of child temperament and behaviour, and concurrent ratings on the Separation Anxiety Test (SAT). Ratings of disorganized attachment on the MCAST show association with Unresolved status on concurrent maternal Adult Attachment Interview (AAI) and with independent teacher ratings of classroom behaviour. Child temperament shows an effect on the style of engagement with the interview but not the attachment ratings. Possible alternative explanations for the phenomena seen are discussed, along with potential applications of the instrument and directions for future research.

Neosho County Community College, is a small college in Kansas. It set out to measure outcomes in every course, program, and in general education. It wound up creating a simple, elegant, inexpensive, faculty-driven system that transformed the college culture from an emphasis on what was being taught to what was being learned.

Many chronic pain patients seem to have difficulty regulating exercise programs and require detailed, precise instructions of what exercises to do as well as their frequency and timing. This may be due to a lack of information concerning the state of the damaged tissues, which may result from inadequate feedback mechanisms for pain, stretch and position. The straight leg raising test was used as a model of this mechanism, and variance in targeting the end-point of this test was measured in patients with chronic pain and in controls. Variance about the end-point in chronic pain patients was more than three times larger than that in controls. Although chronic pain patients were older, there was no correlation between straight leg raising and age. The implications of these findings to the maintenance of the original injury and the importance of this in the maintenance and continuation of chronic pain states are discussed.

We describe a new instrument, using a doll-play vignette completion method, which applies concepts and methodologies from infant and adult attachment research to enable identification and detailed classification of internal representations of attachment relationships in young school-age children. Validation of the Manchester Child Attachment Story Task (MCAST) in a normal population (N = 53) shows good interrater reliability and content validity. Patterns of attachment representation identified show stability over time. Comparisons are made with existing methodologies, and potential applications of the instrument and directions for future research are discussed.

A rapid high-pressure liquid chromatographic assay is described for the quantitative determination of griseofulvin in plasma. An aliquot (25--10 microliter) of plasma was deproteinized by a simple procedure involving the addition of 2.5 volumes of acetonitrile, vortex mixing for a few seconds, and centrifugation for 1 min. The clear supernate, 50 microliter, was injected into the high-pressure liquid chromatograph. A reversed-phase column was used with a mobile phase of distilled water-acetonitrile (1:1) at a flow rate of 2 ml/min and was operated at ambient temperature. A fluorescent detector with an excitation wavelength of 260 nm was employed to monitor the column effluent. Griseofulvin had a retention time of 3.8 min. This procedure yields reproducible results with high sensitivity; plasma concentrations as low as 50 ng/ml can be measured. Several commonly used drugs do not interfere. Analysis of plasma samples collected from a rabbit injected with griseofulvin indicated that the procedure is suitable for pharmacokinetic studies and clinical monitoring of plasma concentrations in patients. Assay turnaround time is less than 6 min. For clinical monitoring of plasma griseofulvin concentrations, a sample volume as small as 10 microliter can be used.