National Research Ethics Service Research Articles

Sleep and motor learning in stroke (SMiLES): a longitudinal study investigating sleep-dependent consolidation of motor sequence learning in the context of recovery after stroke

IntroductionThere is growing evidence that sleep is disrupted after stroke, with worse sleep relating to poorer motor outcomes. It is also widely acknowledged that consolidation of motor learning, a critical...

Efficacy and optimal dose of acetic acid to treat colonised burns wounds: protocol for a pilot randomised controlled trial

IntroductionDespite of recent advancement in the burns wound management, burn wound infection (BWI) is still one of the major cause of burns mortality. Patients who survive their burns injury still...

Development of an artificial intelligence system to identify hypoglycaemia via ECG in adults with type 1 diabetes: protocol for data collection under controlled and free-living conditions

IntroductionHypoglycaemia is a harmful potential complication in people with type 1 diabetes mellitus (T1DM) and can be exacerbated in patients receiving treatment, such as insulin therapies, by the very interventions...

Improving sleep and learning in rehabilitation after stroke, part 2 (INSPIRES2): study protocol for a home-based randomised control trial of digital cognitive behavioural therapy (dCBT) for insomnia

IntroductionConsolidation of motor skill learning, a key component of rehabilitation post-stroke, is known to be sleep dependent. However, disrupted sleep is highly prevalent after stroke and is often associated with...

Genetic Validation of Psoriasis Phenotyping in UK Biobank Supports the Utility of Self-Reported Data and Composite Definitions for Large Genetic and Epidemiological Studies

Genetic Validation of Psoriasis Phenotyping in UK Biobank Supports the Utility of Self-Reported Data and Composite Definitions for Large Genetic and Epidemiological Studies

Observational prospective study of social media, smartphone use and self-harm in a clinical sample of young people: study protocol

IntroductionYoung people are the most frequent users of social media and smartphones and there has been an increasing speculation about the potential negative impacts of their use on mental health....

Minimally invasive surgery for lesser toe deformity: a clinical audit of a proposed treatment algorithm

Objective: There is increasing interest in the performance of minimally invasive foot surgery (MIS); however, limited evidence and treatment algorithms are available to support its use and guide surgical decision-making. The aim of this prospective clinical audit was to report the efficacy of a treatment algorithm used to treat patients presenting with lesser toe deformities using MIS techniques. Methods: A prospective clinical audit of 38 patients who underwent 55 MIS procedures for complex and simple lesser toe deformities was conducted between April 2018 and June 2022. All patients were followed up for a minimum of 12 months post operatively. The audit was conducted following the National Research Ethics Service (NRES) guidelines on clinical audit. Results: Mean pre-operative Visual Analogue Pain (VAS) score was 3.95 with a median of 5.00. The mean post-operative VAS scores improved to 0.23 after six weeks and 0.43 after 12 weeks. A Mann-Whitney U test concluded that this improvement was statistically significant (p<0.05). Conclusion: This algorithm appears effective in treating lesser toe deformities independent of deformity classification, concomitant surgery, gender or whether the surgery was performed in a hospital or private clinical setting. Level of Evidence V; Therapeutic Study; Expert Opinion.

HPB O05 Identifying pathways involved in post-hepatectomy liver failure

Abstract Background The liver is the only visceral organ with regenerative capabilities. This ensures maintenance of the liver-to-bodyweight ratio required for homeostasis. Failure of this mechanism can be fatal. Precipitants to liver failure include infection, drug toxicity and surgical resection. Post-hepatectomy liver failure (PHLF) is associated with small for size syndrome i.e., insufficient post hepatectomy liver volume. The mechanisms by which liver failure occurs on a molecular basis are not yet fully understood, but theories include uncontrolled apoptosis from unrivalled superoxide radical formation causing irreparable oxidative stress. A major research focus of liver regeneration has been modulating the anti-oxidative response to any form of liver injury, but identifying pathways involved in dysfunctional liver regeneration after surgery have yet to be elucidated. Nuclear factor erythoid 2-related factor 2 (Nrf2) is an anti-oxidant transcription factor which promotes liver regeneration. Using a murine partial hepatectomy model, we have shown that pharmacological stimulation of Nrf2 increases the rate of post hepatectomy liver regeneration. Furthermore glutathione (GSH – a downstream target of Nrf2), has been linked with impaired liver regeneration when deficient. In this study, we present data on GSH analysis in liver tissue taken from patients undergoing hepatectomy. Methods Liver parenchyma was collected ex-vivo from patients undergoing liver resection (National Research Ethics Service approval 11/NW/0327). Additionally, isolated RNA from hepatic parenchyma from patients undergoing liver resections was received from collaborators at the Medical University of Vienna, Austria. From liver tissue, protein was isolated using a standard protocol and immunoblotting performed; RNA was isolated using a Monarch RNA preparation kit (NEB, UK), and quantitative PCR was performed. GSH levels in liver tissue were analysed using a standard enzyme recycling method. Relevant targets were identified based on their response to anti-oxidative stress as defined in literature. Primers were designed based on internally validated sequences and antibodies were purchased externally. qPCR was performed using the fluorescent dye, SYBR Green with a ViiA 7 Real-Time PCR system (ThermoFisher). Immunoblotting consisted of transferring separated by weight proteins onto a membrane which was then blocked in 5% milk, with primary and then secondary antibody incubation. Chemiluminescence was applied and bands were visualised using a ChemiDoc (BioRad). Bands were quantified using ImageJ software, and expressed in relation to actin. PHLF was classified according to the ISGLS criteria. Control patients (with no PHLF) were matched for demographic and clinico-pathologic variables (major resection as per the Brisbane criteria). Results Protein expression and GSH expression was measured in liver tissue from 23 patients. Of these, 10 had no PHLF, 4, 3 and 6 were classified as having Grades A, B, and C PHLF respectively. Additional isolated RNA samples yielded 11 with no PHLF, and 14 PHLF (4, 7 and 3, grade A, grade B and grade C, respectively) patients. A GSH assay showed no change in expression between those without PHLF and those with grade A and grade B, but patients who had grade C PHLF had significantly lower levels of GSH (p=0.0040). With respect to gene expression, GCLC, encoding the enzyme glutamate cysteine ligase catalytic subunit, the first rate limiting enzyme of glutathione synthesis, was also significantly lower in patients with grade C compared to those without PHLF of grade A/B (p=0.0206). Protein expression showed significantly reduced levels of GCLC when comparing grade C with patients without PHLF. Furthermore, samples taken at 2 timepoints (pre-parenchymal transection, and then 2 hours later from the resection margin) have shown a decrease in GCLC gene expression in patients who have grade C PHLF. Conclusions Our results have shown that patients who have grade C PHLF (who all died from acute liver failure post-operatively), have significantly lower levels of GSH in liver tissue taken at the time of resection. There is already a weight of evidence showing activation of the transcription factor Nrf2 (encoding GSH) enhances liver regeneration, however this is not without off target effects. Although not known to promote oncogenesis, in vitro studies have shown that persistent overstimulation of Nrf2 promotes cancer cell growth and proliferation, in part by improving the chemoresistance of cancer cells. This effect is theorised to be due to metabolic reprogramming of cancer cells, resulting in elevated levels of metabolic enzymes involved in cell proliferation.Whilst it is appreciated that liver failure is multi-factorial, targeting genes that promote liver regeneration may reduce the risk of failure. Identifying and targeting individual genes which are involved in liver regeneration may help to overcome potentially deleterious off target effects. This data suggests that GSH is a potential therapeutic target reducing the risk of PHLF.

Study protocol for a multicentre comparative diagnostic accuracy study of tools to establish the presence and severity of peripheral arterial disease in people with diabetes mellitus: the DM PAD study

IntroductionPeripheral arterial disease (PAD) is a key risk factor for cardiovascular disease, foot ulceration and lower limb amputation in people with diabetes. Early diagnosis of PAD can enable optimisation of...

Association between individual-level socioeconomic position and incident dementia using UK Biobank data: a prospective study

BackgroundUnder-education and living in poverty are known risk factors for dementia. However, single-variable makers of socioeconomic position (SEP) are often correlated and cannot reflect the overall SEP. We examined association between composite SEP and incident dementia using UK-wide data. MethodsWe leveraged data from the UK Biobank, a nation-wide cohort of half-a-million participants recruited across 22 assessment centres during 2006–10. Participants with data on SEP and without dementia at baseline were included. A composite individual-level metric of SEP (low, medium, or high) was developed through latent class analysis using Stata's gsem command and identified through item-response probabilities based on participants' single socioeconomic factors (ie, education, employment, and household income). Cox proportional hazard models were developed to examine the association between composite SEP and incident dementia after adjusting for age, ethnicity, lifestyle factors (eg, living alone), social interaction (eg, frequency of visits from acquaintances), urbanicity, clinical variables (eg, central obesity), and stratified by sex. As sensitivity test, we repeated our analysis with single socioeconomic factors. Electronic informed consent was obtained from participants at the UK Biobank assessment centres before participations and UK Biobank acquired ethical approval from the National Health Service National Research Ethics Service. FindingsWe included 340 366 adult participants (17 8195 [52·4%] were women and 32 6753 [96·0%] White) aged 38–73 with 3541 incident dementia cases over a mean follow-up period of 12·0 years (SD 1·7). Relative to participants in the highest SEP, those in the medium (HR 1·53 [95% CI 1·33–1·77]; p<0·0001) and low (2·38 [2·05–2·77]; p<0·0001) SEP were associated with higher risks of incident dementia. Sensitivity analyses consistently found higher risks of incident dementia in participants of low educational attainment (HR 1·14 [95% CI 1·03–1·27]; p=0·0107), low household income (HR 2·33 [95% CI 2·03–2·68]; p<0·0001) and being unemployed (HR 1·27 [95% CI 1·11–1·47]; p=0·0008), relative to those of high education, high household income, and being employed, respectively. Limitations of the study include a response of only 503 325 (5·5%) of 9·2 million participants registered with the National Health Service and residual confounding. InterpretationThis study presented a parsimonious approach to construct a composite metric of SEP by employing three key indicators. We found that participants of low SEP were associated with an elevated risk of incident dementia. Socially deprived populations maybe more likely to be exposed to unfavourable psychosocial and environmental stressors that escalate risk of dementia. Our study further strengthens the evidence base for designing policy interventions for at-risk subgroups of lower SEP strata to reduce burdens of dementia. FundingNone.

STXBP1 Stop-Loss Mutation Associated with Complex Early Onset Movement Disorder without Epilepsy.

STXBP1 Stop-Loss Mutation Associated with Complex Early Onset Movement Disorder without Epilepsy.

PHOENIX (Picking up Hidden Osteoporosis Effectively during Normal CT Imaging without additional X-rays): protocol for a randomised, multicentre feasibility study

IntroductionTwo million out of the UK’s 5 million routine diagnostic CT scans performed each year incorporate the thoracolumbar spine or pelvic region. Up to one-third reveal undiagnosed osteoporosis or vertebral...

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality, potentially affecting all treatment outcomes.

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality, potentially affecting all treatment outcomes.

Investigating the uptake, effectiveness and safety of COVID-19 vaccines: protocol for an observational study using linked UK national data

IntroductionThe novel coronavirus SARS-CoV-2, which emerged in December 2019, has caused millions of deaths and severe illness worldwide. Numerous vaccines are currently under development of which a few have now...

Associations of neighbourhood housing density with loneliness and social isolation: a cross-sectional study using UK Biobank data

BackgroundWith increasing urbanisation, the role of urban density on mental wellbeing has gained importance. However, the evidence linking housing density, a fundamental attribute of urban built environment, with loneliness and social isolation has been scarce. We examined associations of neighbourhood housing density with loneliness and social isolation in a UK-wide cohort by using the high-resolution UK Biobank Urban Morphometric Platform (known as UKBUMP) database created by our team. MethodsWe leveraged data from UK Biobank, a cohort of adult participants aged 37–73 years recruited between 2006 and 2010. Neighbourhood housing unit density was objectively measured within a walkable scale of 1km and 2km street catchments of participant's geocoded dwelling (equivalent to approximately a 10-min and 20-min walk). Other built environment factors, including density of public transport, traffic intensity of the nearest street, terrain variability, street-level physical walkability, and greenspace, were also modelled. A series of logistic regression models examined the associations of housing density with loneliness and social isolation after rigorous adjustments for built environment and sociodemographic covariates. Restricted cubic spline models were used to examine non-linear associations. We stratified analyses by sex and age. Ethical approval was received by the UK Biobank from the National Health Service National Research Ethics Service and data access for the study was approved by the UK Biobank Scientific Committee. FindingsOur study comprised 478 265 participants (260 641 [54·5%] of whom were women) with complete data on either outcome and housing density. 281 418 and 291 190 participants remained after full adjustment in models of loneliness and social isolation, respectively. Each 1000 units per km2 increment in housing density within a 1 km residential street catchment was independently associated with an increase of 3·7% (OR 1·037 [95% CI 1·02–1·05], p<0·0001) and 17·4% (1·174 [1·16–1·19], p<0·0001) in odds of loneliness and social isolation after full adjustment. The results remained consistent for a catchment of 2km. A non-linear dose-response was observed. The association between housing density and loneliness was more pronounced among participants aged 50–59 years (participants aged <50 years: OR 1·108 [95% CI 0·97–1.26], p=0·1171; 50–59 years: 1·188 [1·06–1·33], p=0·0023; ≥60 years: 1·114 [0·99–1·25], p=0·0646 for the highest housing density quartile vs the lowest; pinteraction=0.0171) and among men (men: 1·235 [1·12–1·36], p<0·0001; women: 1·051 [0·96–1·16], p=0·3054 for the highest exposure quartile vs the lowest; pinteraction<0·0001). Limitations included the cross-sectional study design, non-representativeness of the UK Biobank base population, and potential residual confounding. InterpretationIn a UK-wide study, we found that increased neighbourhood housing density was associated with increased odds of loneliness and social isolation. High density environments might exacerbate unwanted social contacts, resulting in higher social stress and reduced social connections. With more than 65% of the global population projected to reside in urban areas by 2050, strategic city planning achieved by optimising residential density might constitute a population-wide preventive approach for reducing burdens of loneliness and social isolation. FundingPost-graduate research fellowship of the University of Hong Kong.

Non-linear isocaloric substitution analysis: an example using the UK Biobank prospective cohort study

BackgroundDiet is one of the major modifiable lifestyle risk factors of mortality. When total energy intake is held constant, isocaloric substitution of energy sources can also affect mortality risk. The assumption in conventional methods that this exposure-outcome relationship is linear has been found to be untrue in many scenarios. This paper explores non-linear isocaloric substitution analysis using the UK Biobank data. MethodsThis is a UK Biobank prospective cohort study including 195 658 participants from England, Scotland, and Wales with complete information in at least one dietary questionnaire. Baseline data were collected between 2007 and 2010. Participants who reported non-typical diets were excluded. The exposure variables were intakes of carbohydrates (including sugar, starch, fibre), fat (including monounsaturated, polyunsaturated, and saturated fat) and protein, assessed using a 24 h dietary recall. Prospective all-cause mortality, the outcome variable, was derived from linkage to death records. We did a non-linear isocaloric substitution analysis using penalised cubic splines in Cox proportional hazard models. This analysis provides effect sizes conditional on the current intake, whereas conventional analysis provides an average over the whole range of intake. Analyses were adjusted for sociodemographic and other lifestyle factors. This study was performed under generic ethics approval obtained by UK Biobank from the National Health Service National Research Ethics Service. Written consent was obtained from all participants. FindingsThe majority (187 549) of the participants were White and 55·9% were female. Five of nine of the associations between macronutrient intake (as percentage of total energy, %E) and all-cause mortality were non-linear. Conventional isocaloric substitution showed no difference in mortality risk when replacing sugar with starch. However, the non-linear method revealed that replacing sugar with starch was associated with approximately 10% higher risk per 5%E when the current starch intake was greater than 30% of total energy, and with approximately 7% lower risk per 5%E when current intake was less than 25%. InterpretationWhen the underlying exposure-outcome association is non-linear, a relevant non-linear isocaloric substitution analysis technique should be used. The approach described here could provide more meaningful individualised dietary advice in line with precision public health. FundingNone.

Ethics review, reflective equilibrium and reflexivity.

Background:Research Ethics Committees (RECs) or their equivalent review applications for prospective research with human participants. Reviewers use universally agreed principles i to make decisions about whether prospective health and social care research is ethical. Close attention to understanding how reviewers go about their decision-making work and consider principles in practice is limited.Objective:The study aimed to understand how reviewers made decisions in the contexts of meetings and to understand more about how reviewers approach their work. The purpose of this article is to draw on data and findings and to show how reflective equilibrium as a theoretical frame can (1) deepen understanding of ethics review and (2) permit a reflexive examination of the habitual processes of review.Design and participants:Methods captured the day-to-day work of the RECs. Seventeen applications were heard during eight observations. There were 12 formal interviews with reviewers (n = 12) and with researchers (n = 8) which are not reported on in this article.Ethical considerations:Organisational permission for the study was given by the National Research Ethics Service (NRES) whose functions became part of the Health Research Authority (HRA) during the study. The study was given favourable opinion by the University of Salford's REC (Reference HSCR11/17).Findings:Data were analysed using constructed grounded theory resulting in eight themes which revealed attention to procedure and engagement with applications. Reflective equilibrium was used as a qualitative frame to interpret themes distilling them into three processes at work in review: emotion and intuition; imagination and creative thinking; and intuition and trust.Discussion:Reviewers went back and forth between universal principles and considered these in the contexts of each application using the above processes.Conclusions:Reflective equilibrium offers a coherent and grounded account of review work. Reflexivity in training for reviewers is essential for improving practices. The challenges reflexivity presents can be assisted by using reflective equilibrium as a tool to illuminate tacit review processes.

Mitochondria Are Transferred from Non-Malignant Stroma Cells to Malignant Plasma Cells, Via Tunnelling Nanotubules in a CD38 Dependent Mechanism, a Process That Promotes Myeloma Proliferation

BackgroundSurvival and proliferation of multiple myeloma (MM) is dependent on the bone marrow microenvironment where the malignant plasma cells rely on both glycolysis and oxidative phosphorylation to generate ATP. Recent work by our group and others have shown that malignant cells can acquire mitochondria from benign non-malignant cells in the tumor micro-environment (Marlein at al. 2017 Blood). Furthermore others have shown that CD38 controls mitochondrial transfer between astrocytes and neurons after stroke (Hayakawa et al. 2016 Nature). Therefore, since mitochondria are essential for the survival, proliferation and chemotherapy resistance of leukemia together with the knowledge that CD38 controls mitochondrial transfer in brain injury we hypothesised that mitochondria are transferred to MM from BMSC via CD38 dependent mechanism.MethodsBone marrow containing primary MM cells was obtained from patients following informed consent, in accordance with the Declaration of Helsinki and under approval from the United Kingdom National Research Ethics Service. Malignant plasma cells were purified using magnetic-activated cell sorting with CD138+ microbeads. Primary BMSC were also purified from patient bone marrow, using adherence and characterised using flow cytometry for expression of CD90+, CD73+, CD105+ and CD45-. MM-derived cell lines were obtained from the European Collection of Cell Cultures where they are authenticated by DNA fingerprinting. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining and an in vivo MM NSG xenograft model. Visualisation of tunnelling nanotubes (TNT) was achieved using fixed cell confocal microscopy, quantification of these was carried out by quantification of TNT-anchor points [1]. CD38 expression on primary MM and cell lines was analysed using flow cytometry and qPCR. To inhibit CD38 we used a blocking antibody and lentivirus targeted shRNA. In vivo MM progression in an NSG MM xenograft model was monitored through bioluminescent live animal imaging over time and by overall survival.ResultsFirst we stained BMSC with MitoTracker Green stain and cultured these with primary MM cells. Using flow cytometry and confocal microscopy we detected MitoTracker fluorescence in the MM cells after co-culture, showing that stained mitochondria are transferred from BMSC to MM cells in-vitro. In a human MM cell line into NSG mouse xenograft model, we detect murine mitochondrial DNA in sorted human MM1S and U266 cells post-transplant. Through fixed cell confocal microscopy we found that mitochondria move through TNTs. Using our BMSC with MitoTracker Green stain MM co-culture assay, we found that CD38 knockdown (Kd) of MM1S cells reduced mitochondrial transfer in-vitro. CD38 Kd improved survival in our NSG xenograft model and moreover blocking CD38 expression reduced mitochondrial transfer between BMSC and MM cells in-vivo. Finally the number of tumor derived TNT-anchor points on BMSC is significantly reduced after co-culture with CD38 knockdown MM cells compared to control knockdown MM cells, highlighting that CD38 is important for TNT formation and binding to BMSC.ConclusionHere we show for the first time that mitochondrial transfer occurs between BMSC and malignant plasma cells which supports the growth and proliferation of MM in-vivo and in-vitro. Moreover we report that the mitochondria move through TNTs and that CD38 expression on MM is functionally important for this pro-tumoral mitochondrial transfer to occur. DisclosuresNo relevant conflicts of interest to declare.

PGC1α Driven Mitochondrial Biogenesis within the Bone Marrow Stromal Cells of the Acute Myeloid Leukemia Micro-Environment Is a Pre-Requisite for Mitochondrial Transfer to Leukemic Blasts

BackgroundThe bone marrow (BM) microenvironment is essential for the growth and proliferation of acute myeloid leukemia (AML). Recently we have reported that mitochondria are transferred from BM stromal cells (BMSC) to AML blasts through tunnelling nanotubes (TNTs), a process which supports leukemic proliferation in-vitro and in-vivo. Moreover, we showed that tumor derived NOX2 generates superoxide which provides the stimulus to drive mitochondrial transfer in this way (Marlein at al. 2017 Blood). In the present study we specifically examine the effects of AML derived superoxide on BMSC mitochondrial biogenesis and subsequent mitochondrial transfer to AML.MethodsPrimary AML blasts and BMSC were obtained from patient bone marrow, with informed consent and under approval from the UK National Research Ethics Service. Animal experiments were performed following approval by the UK Home Office and University of East Anglia Animal Welfare and Ethical Review Board. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) activity was analysed using Western blotting and lentiviral-mediated knockdown. Mitochondrial transfer was assessed and quantified in vitro using a MitoTracker green FM staining and flow cytometry. AML derived superoxide was quenched by glutathione and diphenyleneiodonium (DPI). Leukemic proliferation and tumor volume, in an NSG AML xenograft subcutaneous model, were monitored through bioluminescent live animal imaging.ResultsFirst we quantified levels of mitochondrial transfer with and without inhibition of superoxide activity. We found that the anti-oxidants glutathione and DPI significantly reduced levels of mitochondrial transfer from BMSC to AML. We next quantified mitochondrial biogenesis within BMSC, showing that BMSC had increased mitochondrial DNA and increased MitoTracker Green FM staining in response to co-culture with AML. Next we report that this increase in mitochondrial content also occurred in response to hydrogen peroxide, which mimics the effects of AML-derived superoxide. In addition, we show that PGC1α (master regulator of mitochondrial biogenesis) levels in the nucleus of BMSC increased when cultured with AML. Moreover, knockdown of PGC1α inhibited mitochondrial biogenesis in BMSC when cultured with AML or activated with hydrogen peroxide. Furthermore, transfer of mitochondria from BMSC to AML was substantially reduced when BMSC-derived PGC1α was silenced. Finally, we engrafted control or PGC1α knockdown BMSC together with OCI-AML3 cells (AML cell line) into the sub-cutaneous tissue of NSG mice. We found significantly reduced AML tumour volume when the AML was transplanted with the PGC1α knockdown BMSC compared to transplant of AML with the control knockdown stromal cells.ConclusionHere we report that the superoxide generated by AML drives BMSC mitochondrial biogenesis and subsequent mitochondrial transfer between BMSC and leukemic blasts. Targeting this pathway may provide an effective novel therapeutic approach in AML DisclosuresNo relevant conflicts of interest to declare.

Acute Myeloid Leukaemia Induces p16 Driven Senescence in the Bone Marrow Microenvironment to Support Their Proliferation and Survival

BackgroundAcute myeloid leukaemia (AML) occurs most commonly in older people and depends on the bone marrow microenvironment. The aging bone marrow accumulates senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of numerous pro-inflammatory cytokines, chemokines and growth factors, termed the secretary associated senescence profile (SASP). Previously we shown that AML is capable of reprogramming bone marrow stromal cells (BMSC) to support blast survival and proliferation. Here, we hypothesis that AML programs a senescent aging phenotype in the BMSC, generating a SASP in the BMSC which then feedback to promote AML blast proliferation in the bone marrow niche.MethodsWe isolated primary AML cells and BMSC from the bone marrow, with informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). We characterized BMSC by flow cytometry for expression of CD90+, CD73+, CD105+ and CD45, and assayed cytokines and chemokines secreted by primary AML cells, primary BMSC and AML/BMSC co-cultures using a proteomic array. To assess senescence of BMSC, we measured p16 mRNA and protein levels and senescence associated β-Galactosidase activity. We also transplanted syngeneic AML blasts (generated from lineage negative cells transduced with the oncogene MN1) into p16-3MR mice (Demaria, M., et al. 2014 Cell Development), in which the herpes simplex virus thymidine kinase (HSV-TK) moiety of the p16-3MR transgene allows selective killing of senescent p16-expressing cells by the pro-drug ganciclovir (GCV). We also used the NSG xenograft model in which we subcutaneously transplanted primary human AML cells and BMSC (either control and p16-kockdown).ResultsWe report that human AML cells induce BMSC to secrete a cytokine profile (including interleukin-6, interleukin-8, MIP1a and MIP2a). To examine whether AML cells can induce senescence in BMSC in culture, we co-cultured primary AML-derived BMSC with primary AML blasts and assayed for the senescent phenotype. We found that primary AML cells can induce p16 mRNA and protein and increase senescence associated β-Galactosidase activity in the BMSC after 6 days in culture. Furthermore, primary AML cells cultured on p16 knockdown BMSC had reduced survival compared to control wild type BMSC. In vivo we show that MN1 blasts engraft p16-3MR mice and the depletion of p16 positive stromal cells through treating animals with GVC compared to vehicle control significantly increased survival of MN1 engrafted mice. Finally, subcutaneous injection of human AML cells with human p16-KD BMSC significantly decreased tumour volume compared to injection of human AML cells with control-KD BMSC in mouse xenograft model.ConclusionHere we show that the AML blast induces a senescent phenotype in the bone marrow microenvironment which then feeds back to enhance the growth capacity of the AML blast. Targeting the senescent microenvironment may provide a novel therapeutic approach for the treatment of this disease. DisclosuresCampisi:Unity Biotechnology: Equity Ownership.