Publications by authors named "Cheryl L Scudamore"

43 Publications

Research Relevant Background Lesions and Conditions: Ferrets, Dogs, Swine, Sheep, and Goats.

ILAR J 2021 Mar 13. Epub 2021 Mar 13.

Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Animal models provide a valuable tool and resource for biomedical researchers as they investigate biological processes, disease pathogenesis, novel therapies, and toxicologic studies. Interpretation of animal model data requires knowledge not only of the processes/diseases being studied but also awareness of spontaneous conditions and background lesions in the model that can influence or even confound the study results. Species, breed/stock, sex, age, anatomy, physiology, diseases (noninfectious and infectious), and neoplastic processes are model features that can impact the results as well as study interpretation. Here, we review these features in several common laboratory animal species, including ferret, dog (beagle), pig, sheep, and goats.
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http://dx.doi.org/10.1093/ilar/ilab005DOI Listing
March 2021

The occurrence of tarsal injuries in male mice of C57BL/6N substrains in multiple international mouse facilities.

PLoS One 2020 15;15(6):e0230162. Epub 2020 Jun 15.

Mary Lyon Centre, MRC Harwell Institute, Oxfordshire, United Kingdom.

Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in group-housed adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneus-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230162PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295225PMC
August 2020

Loss of Phosphatidylinositol 3-Kinase Activity in Regulatory T Cells Leads to Neuronal Inflammation.

J Immunol 2020 07 15;205(1):78-89. Epub 2020 May 15.

Laboratory of Lymphocyte Signalling and Development, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom;

Class I PI3K enzymes are critical for the maintenance of effective immunity. In T cells, PI3Kα and PI3Kδ are activated by the TCR and costimulatory receptors, whereas PI3Kγ is activated by G protein-coupled chemokine receptors. PI3Kδ is a key regulator of regulatory T (Treg) cell function. PI3K isoform-selective inhibitors are in development for the treatment of diseases associated with immune dysregulation, including chronic inflammatory conditions, cancer, and autoimmune diseases. Idelalisib (PI3Kδ), alpelisib (PI3Kα), duvelisib (PI3Kδ/γ), and copanlisib (pan-PI3K) have recently been approved for use in cancer treatment. Although effective, these therapies often have severe side effects associated with immune dysregulation and, in particular, loss of Treg cells. Therefore, it is important to gain a better understanding of the relative contribution of different PI3K isoforms under homeostatic and inflammatory conditions. Experimental autoimmune encephalitis is a mouse model of T cell-driven CNS inflammation, in which Treg cells play a key protective role. In this study, we show that PI3Kδ is required to maintain normal Treg cell development and phenotype under homeostatic conditions but that loss of PI3Kδ alone in Treg cells does not lead to autoimmunity. However, combined loss of PI3Kα and PI3Kδ signaling resulted in increased experimental autoimmune encephalitis disease severity. Moreover, mice lacking PI3Kα and PI3Kδ in Treg cells developed spontaneous peripheral nerve inflammation. These results show a key role for PI3K signaling in Treg cell-mediated protection against CNS inflammation.
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http://dx.doi.org/10.4049/jimmunol.2000043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311201PMC
July 2020

Modification of an aggressive model of Alport Syndrome reveals early differences in disease pathogenesis due to genetic background.

Sci Rep 2019 12 31;9(1):20398. Epub 2019 Dec 31.

Renal and Vascular Inflammation Section, Department of Medicine, Imperial College, London, W12 0N, UK.

The link between mutations in collagen genes and the development of Alport Syndrome has been clearly established and a number of animal models, including knock-out mouse lines, have been developed that mirror disease observed in patients. However, it is clear from both patients and animal models that the progression of disease can vary greatly and can be modified genetically. We have identified a point mutation in Col4a4 in mice where disease is modified by strain background, providing further evidence of the genetic modification of disease symptoms. Our results indicate that C57BL/6J is a protective background and postpones end stage renal failure from 7 weeks, as seen on a C3H background, to several months. We have identified early differences in disease progression, including expression of podocyte-specific genes and podocyte morphology. In C57BL/6J mice podocyte effacement is delayed, prolonging normal renal function. The slower disease progression has allowed us to begin dissecting the pathogenesis of murine Alport Syndrome in detail. We find that there is evidence of differential gene expression during disease on the two genetic backgrounds, and that disease diverges by 4 weeks of age. We also show that an inflammatory response with increasing MCP-1 and KIM-1 levels precedes loss of renal function.
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http://dx.doi.org/10.1038/s41598-019-56837-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938516PMC
December 2019

Systemic silencing of PHD2 causes reversible immune regulatory dysfunction.

J Clin Invest 2019 06 4;129(9):3640-3656. Epub 2019 Jun 4.

Nuffield Department of Medicine Research Building, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.
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http://dx.doi.org/10.1172/JCI124099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715380PMC
June 2019

Tracking preleukemic cells in vivo to reveal the sequence of molecular events in radiation leukemogenesis.

Leukemia 2018 06 3;32(6):1435-1444. Epub 2018 Mar 3.

Cancer Mechanisms and Biomarkers Group, Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Didcot, OX11 ORQ, UK.

Epidemiological studies have demonstrated an increased leukemia incidence following ionizing radiation exposure, but to date, the target cells and underlying mechanisms of radiation leukemogenesis remain largely unidentified. We engineered a mouse model carrying a different fluorescent marker on each chromosome 2, located inside the minimum deleted region occurring after radiation exposure and recognized as the first leukemogenic event. Using this tailored model, we report that following radiation exposure, more than half of asymptomatic CBA Sfpi1 mice presented with expanding clones of preleukemic hematopoietic cells harboring a hemizygous interstitial deletion of chromosome 2. Moreover, following isolation of preleukemic hematopoietic stem and progenitor cells irradiated in their native microenvironment, we identified the presence of Sfpi1 point mutations within a subpopulation of these preleukemic cells expanding rapidly (increasing from 6% to 55% in 21 days in peripheral blood in one case), hence identifying for the first time the presence of such cells within a living animal. Importantly, we also report a previously undescribed gender difference in the phenotype of the preleukemic cells and leukemia, suggesting a gender imbalance in the radiation-induced leukemic target cell. In conclusion, we provide novel insights into the sequence of molecular events occurring during the (radiation-induced) leukemic clonal evolution.
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http://dx.doi.org/10.1038/s41375-018-0085-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990525PMC
June 2018

Vps34 PI 3-kinase inactivation enhances insulin sensitivity through reprogramming of mitochondrial metabolism.

Nat Commun 2017 11 27;8(1):1804. Epub 2017 Nov 27.

UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD, UK.

Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.
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http://dx.doi.org/10.1038/s41467-017-01969-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703854PMC
November 2017

Oncogenic PIK3CA induces centrosome amplification and tolerance to genome doubling.

Nat Commun 2017 11 24;8(1):1773. Epub 2017 Nov 24.

UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street London, London, WC1E 6DD, UK.

Mutations in PIK3CA are very frequent in cancer and lead to sustained PI3K pathway activation. The impact of acute expression of mutant PIK3CA during early stages of malignancy is unknown. Using a mouse model to activate the Pik3ca hotspot mutation in the heterozygous state from its endogenous locus, we here report that mutant Pik3ca induces centrosome amplification in cultured cells (through a pathway involving AKT, ROCK and CDK2/Cyclin E-nucleophosmin) and in mouse tissues, and increased in vitro cellular tolerance to spontaneous genome doubling. We also present evidence that the majority of PIK3CA mutations in the TCGA breast cancer cohort precede genome doubling. These previously unappreciated roles of PIK3CA mutation show that PI3K signalling can contribute to the generation of irreversible genomic changes in cancer. While this can limit the impact of PI3K-targeted therapies, these findings also open the opportunity for therapeutic approaches aimed at limiting tumour heterogeneity and evolution.
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http://dx.doi.org/10.1038/s41467-017-02002-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701070PMC
November 2017

Radiosensitization by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity.

Mol Cancer Ther 2018 02 24;17(2):381-392. Epub 2017 Aug 24.

CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, United Kingdom.

As the population ages, more elderly patients require radiotherapy-based treatment for their pelvic malignancies, including muscle-invasive bladder cancer, as they are unfit for major surgery. Therefore, there is an urgent need to find radiosensitizing agents minimally toxic to normal tissues, including bowel and bladder, for such patients. We developed methods to determine normal tissue toxicity severity in intestine and bladder , using novel radiotherapy techniques on a small animal radiation research platform (SARRP). The effects of panobinostat on tumor growth delay were evaluated using subcutaneous xenografts in athymic nude mice. Panobinostat concentration levels in xenografts, plasma, and normal tissues were measured in CD1-nude mice. CD1-nude mice were treated with drug/irradiation combinations to assess acute normal tissue effects in small intestine using the intestinal crypt assay, and later effects in small and large intestine at 11 weeks by stool assessment and at 12 weeks by histologic examination. effects of panobinostat were assessed by qPCR and of panobinostat, TMP195, and mocetinostat by clonogenic assay, and Western blot analysis. Panobinostat resulted in growth delay in RT112 bladder cancer xenografts but did not significantly increase acute (3.75 days) or 12 weeks' normal tissue radiation toxicity. Radiosensitization by panobinostat was effective in hypoxic bladder cancer cells and associated with class I HDAC inhibition, and protein downregulation of HDAC2 and MRE11. Pan-HDAC inhibition is a promising strategy for radiosensitization, but more selective agents may be more useful radiosensitizers clinically, resulting in fewer systemic side effects.
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712223PMC
February 2018

Dmp1 Promoter-Driven Diphtheria Toxin Receptor Transgene Expression Directs Unforeseen Effects in Multiple Tissues.

Int J Mol Sci 2016 Dec 26;18(1). Epub 2016 Dec 26.

Skeletal Biology Group, Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK.

Mice harbouring a dentin matrix protein 1 () promoter-driven human diphtheria toxin (DT) receptor () transgene (Tg) have recently been used to attain targeted ablation of osteocytes by diphtheria toxin (DT) treatment in order to define osteocyte function. Use of these Tg mice has asserted mechano- and novel paracrine regulatory osteocyte functions. To explore osteocyte roles fully, we sought to confirm the selectivity of DT effects in these transgenic mice. However, our findings revealed incomplete DT-induced osteocyte ablation, prevalent misexpression, as well as more prominent histopathological DT-induced changes in multiple organs in Tg than in wild-type (WT) littermate mice. Mechanistic evidence for DT action, via prominent regulation of phosphorylation status of elongation factor-2 (EF-2), was also found in many non-skeletal tissues in Tg mice; indicative of direct "off-target" DT action. Finally, very rapid deterioration in health and welfare status in response to DT treatment was observed in these Tg when compared to WT control mice. Together, these data lead us to conclude that alternative models for osteocyte ablation should be sought and caution be exercised when drawing conclusions from experiments using these Tg mice alone.
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http://dx.doi.org/10.3390/ijms18010029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297664PMC
December 2016

The Influence of the CTIP Polymorphism, Q418P, on Homologous Recombination and Predisposition to Radiation-Induced Tumorigenesis (mainly rAML) in Mice.

Radiat Res 2016 Dec 21;186(6):638-649. Epub 2016 Nov 21.

a   Cancer Genetics and Cytogenetics Group, Radiation Effects Department, Centre for Radiation Chemical and Environmental Hazards, Public Health England, Chilton, Didcot, Oxfordshire, OX11 0RQ, United Kingdom.

Exposure to ionizing radiation increases the incidence of acute myeloid leukemia (AML), which has been diagnosed in Japanese atomic bombing survivors, as well as patients treated with radiotherapy. The genetic basis for susceptibility to radiation-induced AML is not well characterized. We previously identified a candidate murine gene for susceptibility to radiation-induced AML (rAML): C-terminal binding protein (CTBP)-interacting protein (CTIP)/retinoblastoma binding protein 8 (RBBP8). This gene is essential for embryonic development, double-strand break (DSB) resection in homologous recombination (HR) and tumor suppression. In the 129S2/SvHsd mouse strain, a nonsynonymous single nucleotide polymorphism (nsSNP) in Ctip, Q418P, has been identified. We investigated the role of Q418P in radiation-induced carcinogenesis and its effect on CTIP function in HR. After whole-body exposure to 3 Gy of X rays, 11 out of 113 (9.7%) 129S2/SvHsd mice developed rAML. Furthermore, 129S2/SvHsd mouse embryonic fibroblasts (MEFs) showed lower levels of recruitment of HR factors, Rad51 and replication protein A (RPA) to radiation-induced foci, compared to CBA/H and C57BL/6 MEFs, isolated from rAML-sensitive and resistant strains, respectively. Mitomycin C and alpha particles induced lower levels of sister chromatid exchanges in 129S2/SvHsd cells compared to CBA/H and C57BL/6. Our data demonstrate that Q418P nsSNP influences the efficiency of CTIP function in HR repair of DNA DSBs in vitro and in vivo, and appears to affect susceptibility to rAML.
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http://dx.doi.org/10.1667/RR14495.1DOI Listing
December 2016

Exploring the Lean Phenotype of Glutathione-Depleted Mice: Thiol, Amino Acid and Fatty Acid Profiles.

PLoS One 2016 27;11(10):e0163214. Epub 2016 Oct 27.

Department of Pharmacology, University of Oxford, Oxford, United Kingdom.

Background: Although reduced glutathione (rGSH) is decreased in obese mice and humans, block of GSH synthesis by buthionine sulfoximine (BSO) results in a lean, insulin-sensitive phenotype. Data is lacking about the effect of BSO on GSH precursors, cysteine and glutamate. Plasma total cysteine (tCys) is positively associated with stearoyl-coenzyme A desaturase (SCD) activity and adiposity in humans and animal models.

Objective: To explore the phenotype, amino acid and fatty acid profiles in BSO-treated mice.

Design: Male C3H/HeH mice aged 11 weeks were fed a high-fat diet with or without BSO in drinking water (30 mmol/L) for 8 weeks. Amino acid and fatty acid changes were assessed, as well as food consumption, energy expenditure, locomotor activity, body composition and liver vacuolation (steatosis).

Results: Despite higher food intake, BSO decreased particularly fat mass but also lean mass (both P<0.001), and prevented fatty liver vacuolation. Physical activity increased during the dark phase. BSO decreased plasma free fatty acids and enhanced insulin sensitivity. BSO did not alter liver rGSH, but decreased plasma total GSH (tGSH) and rGSH (by ~70%), and liver tGSH (by 82%). Glutamate accumulated in plasma and liver. Urine excretion of cysteine and its precursors was increased by BSO. tCys, rCys and cystine decreased in plasma (by 23-45%, P<0.001 for all), but were maintained in liver, at the expense of decreased taurine. Free and total plasma concentrations of the SCD products, oleic and palmitoleic acids were decreased (by 27-38%, P <0.001 for all).

Conclusion: Counterintuitively, block of GSH synthesis decreases circulating tCys, raising the question of whether the BSO-induced obesity-resistance is linked to cysteine depletion. Cysteine-supplementation of BSO-treated mice is warranted to dissect the effects of cysteine and GSH depletion on energy metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163214PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082875PMC
June 2017

Developing a Collaborative Agenda for Humanities and Social Scientific Research on Laboratory Animal Science and Welfare.

PLoS One 2016 18;11(7):e0158791. Epub 2016 Jul 18.

School of Veterinary Medicine, University of Surrey, Guildford, Surrey, United Kingdom.

Improving laboratory animal science and welfare requires both new scientific research and insights from research in the humanities and social sciences. Whilst scientific research provides evidence to replace, reduce and refine procedures involving laboratory animals (the '3Rs'), work in the humanities and social sciences can help understand the social, economic and cultural processes that enhance or impede humane ways of knowing and working with laboratory animals. However, communication across these disciplinary perspectives is currently limited, and they design research programmes, generate results, engage users, and seek to influence policy in different ways. To facilitate dialogue and future research at this interface, we convened an interdisciplinary group of 45 life scientists, social scientists, humanities scholars, non-governmental organisations and policy-makers to generate a collaborative research agenda. This drew on methods employed by other agenda-setting exercises in science policy, using a collaborative and deliberative approach for the identification of research priorities. Participants were recruited from across the community, invited to submit research questions and vote on their priorities. They then met at an interactive workshop in the UK, discussed all 136 questions submitted, and collectively defined the 30 most important issues for the group. The output is a collaborative future agenda for research in the humanities and social sciences on laboratory animal science and welfare. The questions indicate a demand for new research in the humanities and social sciences to inform emerging discussions and priorities on the governance and practice of laboratory animal research, including on issues around: international harmonisation, openness and public engagement, 'cultures of care', harm-benefit analysis and the future of the 3Rs. The process outlined below underlines the value of interdisciplinary exchange for improving communication across different research cultures and identifies ways of enhancing the effectiveness of future research at the interface between the humanities, social sciences, science and science policy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158791PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948886PMC
July 2017

Inactivation of class II PI3K-C2α induces leptin resistance, age-dependent insulin resistance and obesity in male mice.

Diabetologia 2016 07 30;59(7):1503-1512. Epub 2016 Apr 30.

UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD, UK.

Aims/hypothesis: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice.

Methods: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a).

Results: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age.

Conclusions/interpretation: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis.

Access To Research Materials: All reagents are available upon request.
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http://dx.doi.org/10.1007/s00125-016-3963-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901096PMC
July 2016

Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans.

Sci Transl Med 2016 Mar;8(332):332ra43

UCL Cancer Institute, University College London, London WC1E 6BT, UK.

Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.
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http://dx.doi.org/10.1126/scitranslmed.aad9982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973268PMC
March 2016

Inactivation of the Class II PI3K-C2β Potentiates Insulin Signaling and Sensitivity.

Cell Rep 2015 Dec 19;13(9):1881-94. Epub 2015 Nov 19.

UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK. Electronic address:

In contrast to the class I phosphoinositide 3-kinases (PI3Ks), the organismal roles of the kinase activity of the class II PI3Ks are less clear. Here, we report that class II PI3K-C2β kinase-dead mice are viable and healthy but display an unanticipated enhanced insulin sensitivity and glucose tolerance, as well as protection against high-fat-diet-induced liver steatosis. Despite having a broad tissue distribution, systemic PI3K-C2β inhibition selectively enhances insulin signaling only in metabolic tissues. In a primary hepatocyte model, basal PI3P lipid levels are reduced by 60% upon PI3K-C2β inhibition. This results in an expansion of the very early APPL1-positive endosomal compartment and altered insulin receptor trafficking, correlating with an amplification of insulin-induced, class I PI3K-dependent Akt signaling, without impacting MAPK activity. These data reveal PI3K-C2β as a critical regulator of endosomal trafficking, specifically in insulin signaling, and identify PI3K-C2β as a potential drug target for insulin sensitization.
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http://dx.doi.org/10.1016/j.celrep.2015.10.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675724PMC
December 2015

Loss of arylformamidase with reduced thymidine kinase expression leads to impaired glucose tolerance.

Biol Open 2015 Oct 2;4(11):1367-75. Epub 2015 Oct 2.

Mammalian Genetics Unit, Medical Research Council Harwell, Oxford, Oxfordshire OX11 0RD, UK

Tryptophan metabolites have been linked in observational studies with type 2 diabetes, cognitive disorders, inflammation and immune system regulation. A rate-limiting enzyme in tryptophan conversion is arylformamidase (Afmid), and a double knockout of this gene and thymidine kinase (Tk) has been reported to cause renal failure and abnormal immune system regulation. In order to further investigate possible links between abnormal tryptophan catabolism and diabetes and to examine the effect of single Afmid knockout, we have carried out metabolic phenotyping of an exon 2 Afmid gene knockout. These mice exhibit impaired glucose tolerance, although their insulin sensitivity is unchanged in comparison to wild-type animals. This phenotype results from a defect in glucose stimulated insulin secretion and these mice show reduced islet mass with age. No evidence of a renal phenotype was found, suggesting that this published phenotype resulted from loss of Tk expression in the double knockout. However, despite specifically removing only exon 2 of Afmid in our experiments we also observed some reduction of Tk expression, possibly due to a regulatory element in this region. In summary, our findings support a link between abnormal tryptophan metabolism and diabetes and highlight beta cell function for further mechanistic analysis.
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http://dx.doi.org/10.1242/bio.013342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728350PMC
October 2015

Recommendations for minimum information for publication of experimental pathology data: MINPEPA guidelines.

J Pathol 2016 Jan 28;238(2):359-67. Epub 2015 Nov 28.

Centre for Comparative Pathology, University of Edinburgh, Edinburgh, UK.

Animal models are essential research tools in modern biomedical research, but there are concerns about their lack of reproducibility and the failure of animal data to translate into advances in human medical therapy. A major factor in improving experimental reproducibility is thorough communication of research methodologies. The recently published ARRIVE guidelines outline basic information that should be provided when reporting animal studies. This paper builds on ARRIVE by providing the minimum information needed in reports to allow proper assessment of pathology data gathered from animal tissues. This guidance covers aspects of experimental design, technical procedures, data gathering, analysis, and presentation that are potential sources of variation when creating morphological, immunohistochemical (IHC) or in situ hybridization (ISH) datasets. This reporting framework will maximize the likelihood that pathology data derived from animal experiments can be reproduced by ensuring that sufficient information is available to allow for replication of the methods and facilitate inter-study comparison by identifying potential interpretative confounders.
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http://dx.doi.org/10.1002/path.4642DOI Listing
January 2016

¹H NMR metabolic profiling of plasma reveals additional phenotypes in knockout mouse models.

J Proteome Res 2015 May 20;14(5):2036-45. Epub 2015 Apr 20.

‡Institute of Hepatology, Foundation for Liver Research, 69-75 Chenies Mews, London WC1E 6HX, United Kingdom.

The International Mouse Phenotyping Consortium program has been established to ascribe biological functions to systematically knocked-out (KO) genes by in vivo and ex vivo phenotyping. The plasma clinical chemistry screen includes an assessment of liver, kidney, and bone function and provides a basic lipid profile and histopathology reports on 32 tissues. We report on the inclusion of plasma analysis by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. (1)H NMR spectroscopy data are summarized from 116 running baseline controls with 18 homozygous and 2 heterozygous KO mouse lines along with wild-type controls (typically n = 7 per gender). For the baseline group, the intersample variation of (1)H NMR glucose measurement was 12%, and the (1)H NMR spectroscopy data were influenced by gender and feeding status. There were good correlations between the clinical chemistry and the (1)H NMR spectroscopy measurements for glucose, triglycerides, and HDL cholesterol. Significant differences were observed in two KO lines, Agl (MGI: 1924809) and Bbs5 (MGI: 1919819), by (1)H NMR spectroscopy, clinical chemistry, and histopathology. In a further two KO lines, Elmod1 (MGI: 3583900) and Emc10 (MGI: 1916933), (1)H NMR metabolic differences were observed, but no other ex vivo changes were detected. In the remaining 16 lines, no ex vivo abnormal phenotypes were observed. Plasma (1)H NMR spectroscopy can therefore provide a novel perspective on the function of knocked-out genes.
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http://dx.doi.org/10.1021/pr501039kDOI Listing
May 2015

Pharmacological inhibition of FTO.

PLoS One 2015 1;10(4):e0121829. Epub 2015 Apr 1.

MRC Harwell, Harwell Oxford Campus, Oxfordshire, Oxford, OX11 0RD, United Kingdom.

In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function in vitro and in vivo as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate in vitro. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO's demethylase activity could be therapeutically useful for the treatment of obesity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121829PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382163PMC
April 2016

Histopathological analysis of the respiratory tract.

Curr Protoc Mouse Biol 2014 Dec 11;4(4):181-96. Epub 2014 Dec 11.

Mary Lyon Centre, MRC Harwell, United Kingdom.

The basic anatomy of the mouse respiratory system is similar to that of other mammals and can be usefully examined under the light microscope in phenotyping studies, inhalation toxicity studies, and studies involving mouse models of human disease. In many studies, however, only the lungs are examined, leaving the possibility that phenotypic information from the majority of the conducting airways is lost. This unit provides standard approaches for tissue collection at necropsy and subsequent selection of a range of respiratory tissues for histological and pathological analysis. The major anatomical features to be found in each section are highlighted, and potential artifacts and methods to avoid them are discussed.
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http://dx.doi.org/10.1002/9780470942390.mo140094DOI Listing
December 2014

Acquiring, recording, and analyzing pathology data from experimental mice: an overview.

Curr Protoc Mouse Biol 2014 Mar 21;4(1):1-10. Epub 2014 Mar 21.

Mary Lyon Centre, MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, United Kingdom.

Pathology is often underutilized as an end point in mouse studies in academic research because of a lack of experience and expertise. The use of traditional pathology techniques including necropsy and microscopic analysis can be useful in identifying the basic processes underlying a phenotype and facilitating comparison with equivalent human diseases. This overview aims to provide a guide and reference to the acquisition, recording, and analysis of high-quality pathology data from experimental mice in an academic research setting.
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http://dx.doi.org/10.1002/9780470942390.mo130200DOI Listing
March 2014

Triggered doxorubicin release in solid tumors from thermosensitive liposome-peptide hybrids: Critical parameters and therapeutic efficacy.

Int J Cancer 2015 Aug 5;137(3):731-43. Epub 2015 Feb 5.

Nanomedicine Lab, Institute of Inflammation and Repair, Manchester Cancer Research Centre, Faculty of Medical & Human Sciences, University of Manchester, United Kingdom.

Temperature-sensitive vesicles designed by inclusion of leucine zipper peptides within a lipid bilayer (Lp-Peptide hybrids) encapsulating Doxorubicin (DOX) have been reported. Intravenous administration of these constructs prolonged blood circulation kinetics and increased tumor accumulation in vivo with local mild hyperthermia. In this study, the biological activity of the DOX-loaded Lp-Peptide hybrid vesicles was further investigated at the cellular level and in vivo compared to lysolipid-containing temperature-sensitive liposomes (LTSL) and traditional temperature-sensitive liposomes. Lp-Peptide vesicles were not toxic to cell cultures at 37°C, while effective cancer cell toxicity was observed after 1 hr of heating at 42°C. The activity of Lp-Peptide vesicles in vivo was studied using two different heating protocols to obtain tumor intravascular or interstitial drug release. Lp-Peptide vesicle treatment allowing intravascular DOX release showed equally effective tumor growth retardation and survival to that of LTSL treatment. The Lp-Peptide vesicles also offered therapeutic responses using the alternative heating protocol to maximise drug release within the tumor interstitium. Matching the drug release kinetics of temperature-sensitive vesicles with the heating protocol applied is considered the most critical factor to determine therapeutic efficacy in the clinical translation of such modalities.
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http://dx.doi.org/10.1002/ijc.29430DOI Listing
August 2015

Dual-action combination therapy enhances angiogenesis while reducing tumor growth and spread.

Cancer Cell 2015 Jan;27(1):123-37

Centre for Tumor Biology, Barts Cancer Institute-a CR-UK Centre of Excellence, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK. Electronic address:

Increasing chemotherapy delivery to tumors, while enhancing drug uptake and reducing side effects, is a primary goal of cancer research. In mouse and human cancer models in vivo, we show that coadministration of low-dose Cilengitide and Verapamil increases tumor angiogenesis, leakiness, blood flow, and Gemcitabine delivery. This approach reduces tumor growth, metastasis, and minimizes side effects while extending survival. At a molecular level, this strategy alters Gemcitabine transporter and metabolizing enzyme expression levels, enhancing the potency of Gemcitabine within tumor cells in vivo and in vitro. Thus, the dual action of low-dose Cilengitide, in vessels and tumor cells, improves chemotherapy efficacy. Overall, our data demonstrate that vascular promotion therapy is a means to improve cancer treatment.
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http://dx.doi.org/10.1016/j.ccell.2014.10.015DOI Listing
January 2015

Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer.

Nature 2014 06 11;510(7505):407-411. Epub 2014 Jun 11.

UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street London WC1E 6DD, UK.

Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8(+) cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.
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http://dx.doi.org/10.1038/nature13444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501086PMC
June 2014

A simplified necropsy technique for mice: making the most of unscheduled deaths.

Lab Anim 2014 Oct 29;48(4):342-4. Epub 2014 May 29.

Mary Lyon Centre, MRC Harwell, Oxfordshire, UK.

Sudden or unexpected deaths in experimental mice can be potential causes of lost data or lost opportunities to diagnose health problems. In small animal units and in particular out of normal working hours there may not be the time or expertise available to attempt a careful dissection on unscheduled mortalities or moribund animals. This paper outlines a robust and easy necropsy technique which can be easily learnt, uses minimal equipment and can be used to implement the 3Rs (replacement, refinement and reduction) by maximizing the amount of information gained from experimental animals.
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http://dx.doi.org/10.1177/0023677214536555DOI Listing
October 2014

Integrating pathology into human disease modelling--how to eat the elephant.

Dis Model Mech 2014 May;7(5):495-7

Mary Lyon Centre, MRC Harwell, Oxfordshire, OX11 0RD, UK.

Mouse models are increasingly being used for the study of human disease, and the generation and functional characterisation of new models is underpinned by high-throughput phenotyping consortia such as the International Mouse Phenotyping Consortium. A new study by Adissu and colleagues, published in Disease Models & Mechanisms, demonstrates the usefulness of histopathology in providing corroborative information and uncovering novel phenotypes in genetically modified mice in a high-throughput screen. Although pathology is recognised as a valuable tool to enhance our understanding of animal disease models, it has also been systematically under-resourced. This Editorial aims to highlight ways in which the gap between the usefulness of pathology and its perceived inaccessibility can be addressed by considering pragmatic solutions for planning, resourcing and accessing pathology expertise. The role of funding agencies, academic centres and journals in ensuring that the value of pathology is fully recognised and is adequately supported and funded is also discussed.
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http://dx.doi.org/10.1242/dmm.016394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007400PMC
May 2014

Targeting of Slc25a21 is associated with orofacial defects and otitis media due to disrupted expression of a neighbouring gene.

PLoS One 2014 18;9(3):e91807. Epub 2014 Mar 18.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

Homozygosity for Slc25a21(tm1a(KOMP)Wtsi) results in mice exhibiting orofacial abnormalities, alterations in carpal and rugae structures, hearing impairment and inflammation in the middle ear. In humans it has been hypothesised that the 2-oxoadipate mitochondrial carrier coded by SLC25A21 may be involved in the disease 2-oxoadipate acidaemia. Unexpectedly, no 2-oxoadipate acidaemia-like symptoms were observed in animals homozygous for Slc25a21(tm1a(KOMP)Wtsi) despite confirmation that this allele reduces Slc25a21 expression by 71.3%. To study the complete knockout, an allelic series was generated using the loxP and FRT sites typical of a Knockout Mouse Project allele. After removal of the critical exon and neomycin selection cassette, Slc25a21 knockout mice homozygous for the Slc25a21(tm1b(KOMP)Wtsi) and Slc25a21(tm1d(KOMP)Wtsi) alleles were phenotypically indistinguishable from wild-type. This led us to explore the genomic environment of Slc25a21 and to discover that expression of Pax9, located 3' of the target gene, was reduced in homozygous Slc25a21(tm1a(KOMP)Wtsi) mice. We hypothesize that the presence of the selection cassette is the cause of the down regulation of Pax9 observed. The phenotypes we observed in homozygous Slc25a21(tm1a(KOMP)Wtsi) mice were broadly consistent with a hypomorphic Pax9 allele with the exception of otitis media and hearing impairment which may be a novel consequence of Pax9 down regulation. We explore the ramifications associated with this particular targeted mutation and emphasise the need to interpret phenotypes taking into consideration all potential underlying genetic mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091807PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958370PMC
December 2014

An N-ethyl-N-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess.

Endocrinology 2014 Mar 3;155(3):908-22. Epub 2013 Dec 3.

Mammalian Genetics Unit (L.B., C.T.E., R.A.H., S.D.M.B., R.D.C.) and Mary Lyon Centre (C.L.S., T.A.H.), Medical Research Council Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, United Kingdom; Academic Endocrine Unit (C.T.E., M.A.N., R.A.H., F.M.H., R.V.T.), Nuffield Department of Clinical Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford OX3 7LE, United Kingdom; The Mellanby Centre for Bone Research (H.E., D.L.), Department of Human Metabolism, University of Sheffield, Sheffield S10 2RX, United Kingdom; Wellcome Trust Sanger Institute (C.P.), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom; Norwich Medical School (W.D.F.), University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom; Garvan Institute of Medical Research (P.I.C.), Musculoskeletal Medicine Division, University of New South Wales, Sydney 2010, Australia; and University of Queensland Diamantina Institute (M.A.B.), Princess Alexandra Hospital, University of Queensland, Brisbane 4102, Australia.

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, may be due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh(-120/+) mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh(-120/+) mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh(-120/+) mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh(-120/+) mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.
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http://dx.doi.org/10.1210/en.2013-1247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192286PMC
March 2014