Transcription factor NFE2L1 decreases in glomerulonephropathies after podocyte damage

Funding: This work was supported by NHS Lothian. This project received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 101017453 as part of the KATY project, and from NuCana plc.Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.Publisher PDFPeer reviewe

Dynamic modulation of phosphoprotein expression in ovarian cancer xenograft models

The authors thank Medical Research Scotland and the Scottish Funding Council. This work was su pported by Medical Research Scotland [FRG353 to V.A.S.]; the FP7 -­‐ Directorate -­‐ General for Research and Innovation of the European Commission [EU HEALTH -­‐ F4 -­‐ 2012 -­‐ 305033 to Coordinating Action Systems Medicine -­‐ D.J.H.]; the Chief Scientist Office of Scotland [D.J.H.], the Scottish Funding Council [D.J.H. and S.P.L.]. Health Canada Scholarship (Indspire) [KEF], Scottish Overseas Research Student Award Scheme (University of Edinburgh)[KEF] and the Three Fires Award (Wikwemikong Board of Education)[KEF].Background: The dynamic changes that occur in protein expression after treatment of a cancer in vivo are poorly described. In this study we measure the effect of chemotherapy over time on the expression of a panel of proteins in ovarian cancer xenograft models. The objective was to identify phosphoprotein and other protein changes indicative of pathway activation that might link with drug response. Methods: Two xenograft models, platinum-responsive OV1002 and platinum-unresponsive HOX424, were used. Treatments were carboplatin and carboplatin-paclitaxel. Expression of 49 proteins over 14 days post treatment was measured by quantitative immunofluorescence and analysed by AQUA . Results: Carboplatin treatment in the platinum-sensitive OV1002 model triggered up-regulation of cell cycle, mTOR and DDR pathways, while at late time points WNT, invasion , EMT and MAPK pathways were modulated. Estrogen receptor-alpha (ESR1) and ERBB pathways were down-regulated early, within 24h from treatment administration. Combined carboplatin-paclitaxel treatment triggered a more extensive response in the OV1002 model modulating expression of 23 of 49 proteins. Therefore the cell cycle and DDR pathways showed similar or more pronounced changes than with carboplatin alone . In addition to expression of pS6 and pERK increasing, components of the AKT pathway were modulated with pAKT increasing while its regulator PTEN was down-regulated early. WNT signaling, EMT and invasion markers were modulated at later time points. Additional pathways were also observed with the NFκB and JAK/STAT pathways being up-regulated. ESR1 was down-regulated as was HER4, while further protein members of the ERB B pathway were upregulated late. By contrast, in the carboplatin-unresponsive HOX 424 xenograft, carboplatin only modulated expression of MLH1 while carboplatin-paclitaxel treatment modulated ESR1 and pMET.Publisher PDFPeer reviewe

YAP translocation precedes cytoskeletal rearrangement in podocyte stress response : a podometric investigation of diabetic nephropathy

KH was funded by a University of St Andrews 600th Anniversary Ph.D. scholarship. ME and DH were supported by NHS Lothian.Podocyte loss plays a pivotal role in the pathogenesis of glomerular disease. However, the mechanisms underlying podocyte damage and loss remain poorly understood. Although detachment of viable cells has been documented in experimental Diabetic Nephropathy, correlations between reduced podocyte density and disease severity have not yet been established. YAP, a mechanosensing protein, has recently been shown to correlate with glomerular disease progression, however, the underlying mechanism has yet to be fully elucidated. In this study, we sought to document podocyte density in Diabetic Nephropathy using an amended podometric methodology, and to investigate the interplay between YAP and cytoskeletal integrity during podocyte injury. Podocyte density was quantified using TLE4 and GLEPP1 multiplexed immunofluorescence. Fourteen Diabetic Nephropathy cases were analyzed for both podocyte density and cytoplasmic translocation of YAP via automated image analysis. We demonstrate a significant decrease in podocyte density in Grade III/IV cases (124.5 per 106 μm3) relative to Grade I/II cases (226 per 106 μm3) (Student’s t-test, p<0.001), and further show that YAP translocation precedes cytoskeletal rearrangement following injury. Based on these findings we hypothesize that a significant decrease in podocyte density in late grade Diabetic Nephropathy may be explained by early cytoplasmic translocation of YAP.Publisher PDFPeer reviewe

Use of high-plex data reveals novel insights into the tumour microenvironment of clear cell renal cell carcinoma

This work was supported by Medical Research Scotland (MRS), NHS Lothian, NanoStringTechnologies, and the Industrial Centre for AI Research in Digital Diagnostics (iCAIRD) which is funded by Innovate UK on behalf of UK Research and Innovation (UKRI) [project number: 104690].Although Immune Checkpoint Inhibitors (ICIs) have significantly improved the oncological outcomes, about one third of patients affected by Clear Cell Renal Cell Carcinoma (ccRCC) still experience recurrence. Current prognostic algorithms like the Leibovich Score (LS) rely on morphological features manually assessed by pathologists, and are therefore subject to bias. Moreover, these tools do not consider the heterogeneous molecular milieu present in the Tumour Microenvironment (TME), which may have prognostic value. We systematically developed a semi-automated method to investigate 62 markers and their combinations in 150 primary ccRCCs using multiplex Immunofluorescence (mIF), NanoString GeoMx® Digital Spatial Profiling (DSP) and Artificial Intelligence (AI)-assisted image analysis in order to find novel prognostic signatures and investigate their spatial relationship. We found that coexpression of Cancer Stem Cell (CSC) and Epithelial-to-Mesenchymal Transition (EMT) markers such as OCT4 and ZEB1 are indicative of poor outcome. OCT4 and the immune markers CD8, CD34 and CD163 significantly stratified patients at intermediate LS. Furthermore, augmenting the LS with OCT4 and CD34 improved patient stratification by outcome. Our results support the hypothesis that combining molecular markers has prognostic value and can be integrated with morphological features to improve risk stratification and personalised therapy. To conclude, GeoMx® DSP and AI image analysis are complementary tools providing high multiplexing capability required to investigate the TME of ccRCC, while reducing observer bias.Publisher PDFPeer reviewe

Genome-scale CRISPR/Cas9 screen determines factors modulating sensitivity to ProTide NUC-1031

A.S. is the recipient of a Medical Research Scotland PhD Studentship awarded to P.A.R. Edinburgh Genomics is partly supported through core grants from Natural Environment Research Council (R8/H10/56), Medical Research Council (MR/K001744/1) and Biotechnological and Biological Research Council (BB/J004243/1). Publication of this article was funded in part by the University of St Andrews Open Access Publishing Fund.Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031.Publisher PDFPeer reviewe

Safety and feasibility of countering neurological impairment by intravenous administration of autologous cord blood in cerebral palsy

<p>Abstract</p> <p>Backgrounds</p> <p>We conducted a pilot study of the infusion of intravenous autologous cord blood (CB) in children with cerebral palsy (CP) to assess the safety and feasibility of the procedure as well as its potential efficacy in countering neurological impairment.</p> <p>Methods</p> <p>Patients diagnosed with CP were enrolled in this study if their parents had elected to bank their CB at birth. Cryopreserved CB units were thawed and infused intravenously over 10~20 minutes. We assessed potential efficacy over 6 months by brain magnetic resonance imaging (MRI)-diffusion tensor imaging (DTI), brain perfusion single-photon emission computed tomography (SPECT), and various evaluation tools for motor and cognitive functions.</p> <p>Results</p> <p>Twenty patients received autologous CB infusion and were evaluated. The types of CP were as follows: 11 quadriplegics, 6 hemiplegics, and 3 diplegics. Infusion was generally well-tolerated, although 5 patients experienced temporary nausea, hemoglobinuria, or urticaria during intravenous infusion. Diverse neurological domains improved in 5 patients (25%) as assessed with developmental evaluation tools as well as by fractional anisotropy values in brain MRI-DTI. The neurologic improvement occurred significantly in patients with diplegia or hemiplegia rather than quadriplegia.</p> <p>Conclusions</p> <p>Autologous CB infusion is safe and feasible, and has yielded potential benefits in children with CP.</p

Tissue-specific immunopathology in fatal COVID-19

Funding: Inflammation in COVID-19: Exploration of Critical Aspects of Pathogenesis (ICECAP) receives funding and support from the Chief Scientist Office (RapidResearch in COVID-19 programme [RARC-19] funding call, “Inflammation in Covid-19: Exploration of Critical Aspects of Pathogenesis; COV/EDI/20/10” to D.A.D., C.D.L., C.D.R., J.K.B., and D.J.H.), LifeArc (through the University of Edinburgh STOPCOVID funding award to K.D., D.A.D., and C.D.L.), UK Research and Innovation (UKRI) (Coronavirus Disease [COVID-19] Rapid Response Initiative; MR/V028790/1 to C.D.L., D.A.D., and J.A.H.), and Medical Research Scotland (CVG-1722-2020 to D.A.D., C.D.L., C.D.R., J.K.B., and D.J.H.). C.D.L. is funded by a Wellcome Trust Clinical Career Development Fellowship(206566/Z/17/Z). J.K.B. and C.D.R. are supported by the Medical Research Council (grant MC_PC_19059) as part of the International Severe AcuteRespiratory Infection Consortium Coronavirus Clinical Characterisation Consortium (ISARIC-4C). D.J.H., I.H.U., and M.E. are supported by the Industrial Centre for Artificial Intelligence Research in Digital Diagnostics. S.P. is supported by Kidney Research UK, and G.T. is supported by the Melville Trust for the Cure and Care of Cancer. Identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and sequencing work was supported by theU.S. Food and Drug Administration grant HHSF223201510104C (“Ebola Virus Disease: correlates of protection, determinants of outcome and clinicalmanagement”; amended to incorporate urgent COVID-19 studies) and contract 75F40120C00085 (“Characterization of severe coronavirus infection inhumans and model systems for medical countermeasure development and evaluation”; awarded to J.A.H.). J.A.H. is also funded by the Centre of Excellence in Infectious Diseases Research and the Alder Hey Charity. R.P.-R. is directly supported by the Medical Research Council Discovery Medicine North Doctoral Training Partnership. The group of J.A.H. is supported by the National Institute for Health Research Health Protection Research Unit in Emerging and Zoonotic Infections at the University of Liverpool in partnership with Public Health England and in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.Rationale: In life-threatening Covid-19, corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of SARS-CoV-2 or an independent immunopathologic process is unknown. Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses, and the relationships between viral presence, inflammation, and organ injury. Methods: Tissue was acquired from eleven detailed post-mortem examinations. SARS-CoV-2 organotropism was mapped by multiplex PCR and sequencing, with cellular resolution achieved by in situ viral spike protein detection. Histological evidence of inflammation was quantified from 37 anatomical sites, and the pulmonary immune response characterized by multiplex immunofluorescence. Measurements and main results: Multiple aberrant immune responses in fatal Covid-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein, both between and within tissues. An arteritis was identified in the lung, which was further characterised as a monocyte/myeloid-rich vasculitis, and occurred along with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticulo-endothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues. Conclusions: Tissue-specific immunopathology occurs in Covid-19, implicating a significant component of immune-mediated, virus-independent immunopathology as a primary mechanism in severe disease. Our data highlight novel immunopathological mechanisms, and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma cell responses as well as promoting pathogen tolerance in Covid-19.Publisher PDFPeer reviewe

Computational image analysis in clear cell renal cell carcinoma

Background: In the UK, kidney cancer is the most lethal urologic cancer whose major subtype is renal cell carcinoma (RCC). Surgical resection is the first line of the treatment for renal cell carcinoma (RCC). A number of different integrated staging systems such as UISS, SSIGN and Leibovich score, has been introduced and utilised in clinic as a prognostic tool or as an inclusion criterion of clinical trials. Among them, Leibovich score has been widely utilised in the UK to predict the likelihood of disease free survival for clear cell renal cell carcinoma (ccRCC). However, its prediction rate of disease relapse free for 5 years after surgery varies from 97% (low risk) to 31% (high risk) which might lead to inclusion of some ccRCC patients, who would not recur, into a clinical trial. Therefore, we aim to improve the prediction power of recurrence free (specificity) in localised clear cell renal cell carcinoma (ccRCC) patients, by either modifying Leibovich score with more precise and accurate measurement of ccRCC nuclear morphological features or by improving currently available Leibovich score with the features measured from the chromatin marker colocalisation status, chromatin marker Haralick texture features, and the tumour microenvironment using computational image analysis. Methods: To modify Leibovich score by replacing manual Fuhrman’s nuclear grade with the computational image analysis measurement of the nuclear morphological features, digitised images from haematoxylin and eosin stained slides were utilised. For the chromatin marker (H3K9me3, H3K4me3 and HP1α) colocalisation analysis, Haralick texture analysis and tumour microenvironment marker (CD105 and CD3) analysis, the multiplexed immunofluorescence (IF) was performed and IF images were utilised. The image analysis was performed by using Definiens Tissue studio® (Definiens AG, Munich, Germany) and the Developer platform. Moreover, a novel statistical model was developed using AUCp (partial area under the curve) function in R studio, which defines the range of specificity between 1 and 0.8 on the basis of the binomial GLM (Generalised linear model) framework with GAM-SALSA (Generalised Additive Models - Spatially adaptive local smoothing algorithm) as a calibration tool. In order to avoid the overfitting problem, 5-fold cross validation with 100 times repetition was also added in the analysis. Results: Firstly, our statistical model replaced Fuhrman’s nuclear grade with ‘mean perimeter’, which was named ‘Modified Leibovich algorithm’. The modified Leibovich algorithm improved its overall specificity 0.86 (80 out of 93 cases) from 0.76 (71 out of 93) from the classic L score in the Scottish training cohort. In particular, the most increase in specificity was seen in Leibovich score 5 and 6, which were 57% and 40%, respectively. The modified Leibovich algorithm also increased overall specificity up to 0.94 (141 out of 150 cases), compared to the classic (original) Leibovich score whose specificity was 0.84 (126 out of 150 cases) in a Singaporean validation cohort. Moreover, specificity was dramatically increased in Leibovich score 5 from 0% to 92%. Secondly, the chromatin marker colocalisation feature significantly improved the specificity of the classic and partial Leibovich score up to 0.98 in overall. In particular, the specificity of the cases in Leibovich score 4, 5 and 6 was improved up to 100%. However, the chromatin marker Haralick texture features did not improve the specificity of the classic and partial Leibovich score as much as the chromatin marker colocalisation features. Thirdly, the tumour microenvironment features such as the density and the spatial distances of CD105 positive blood vessels and CD3 positive mature T lymphocytes augmented the specificity of the classic and partial Leibovich score up to 0.93. In particular, it improved the specificity up to 92% in Leibovich score 5 compared to the classic Leibovich score. Conclusions: Computational image analysis enabled to measure such various ranges of features not only in tumour cells, but also in tumour microenvironment. In this study, ccRCC tumour cell nuclear morphological features, chromatin marker colocalisation feature, chromatin Haralick texture features and tumour microenvironment features were visualised by the multiplexed immunofluorescence and measured by the Tissue studio and developer software: this cannot be done manually. The data from this analysis significantly augmented the specificity of the currently available prognostic tool, Leibovich score, in clinic. In particular, the very last final model developed by combining features of the chromatin marker colocalisation (‘Average.HP1a.Intensity..H3K4_HP1a.Overlap.’ and ‘Manders.Coefficient.Nuclei..H3K4.HP1a..M2’) and tumour microenvironment (‘Mean.CD3.Area.percentage.of.All.Tissue’ and ‘Mean.No..of.CD105’) selected along with the classic Leibovich score, predicted 100% (93 out of 93 cases) correctly the cases which did not experience the disease recurrence within 5 years after surgery, while the classic Leibovich score predicted 76% (71 out of 93 cases) correctly. This could have prevented 22 ccRCC patients not only to get unpleasant and unnecessary treatment after nephrectomy, but also helped them having been free from the fear of disease recurrence

MultiPathGAN:structure preserving stain normalization using unsupervised multi-domain adversarial network with perception loss

Histopathology relies on the analysis of microscopic tissue images to diagnose disease. A crucial part of tissue preparation is staining whereby a dye is used to make the salient tissue components more distinguishable. However, differences in laboratory protocols and scanning devices result in significant confounding appearance variation in the corresponding images. This variation increases both human error and the inter-rater variability, as well as hinders the performance of automatic or semi-automatic methods. In the present paper we introduce an unsupervised adversarial network to translate (and hence normalize) whole slide images across multiple data acquisition domains. Our key contributions are: (i) an adversarial architecture which learns across multiple domains with a single generator-discriminator network using an information flow branch which optimizes for perceptual loss, and (ii) the inclusion of an additional feature extraction network during training which guides the transformation network to keep all the structural features in the tissue image intact. We: (i) demonstrate the effectiveness of the proposed method firstly on H&amp;E slides of 120 cases of kidney cancer, as well as (ii) show the benefits of the approach on more general problems, such as flexible illumination based natural image enhancement and light source adaptation.</p