Publications by authors named "Jeffrey I Everitt"

48 Publications

Radiologic-pathologic analysis of increased ethanol localization and ablative extent achieved by ethyl cellulose.

Sci Rep 2021 Oct 19;11(1):20700. Epub 2021 Oct 19.

Department of Biomedical Engineering, Duke University, Durham, NC, USA.

Ethanol provides a rapid, low-cost ablative solution for liver tumors with a small technological footprint but suffers from uncontrolled diffusion in target tissue, limiting treatment precision and accuracy. Incorporating the gel-forming polymer ethyl cellulose to ethanol localizes the distribution. The purpose of this study was to establish a non-invasive methodology based on CT imaging to quantitatively determine the relationship between the delivery parameters of the EC-ethanol formulation, its distribution, and the corresponding necrotic volume. The relationship of radiodensity to ethanol concentration was characterized with water-ethanol surrogates. Ex vivo EC-ethanol ablations were performed to optimize the formulation (n = 6). In vivo ablations were performed to compare the optimal EC-ethanol formulation to pure ethanol (n = 6). Ablations were monitored with CT and ethanol distribution volume was quantified. Livers were removed, sectioned and stained with NADH-diaphorase to determine the ablative extent, and a detailed time-course histological study was performed to assess the wound healing process. CT imaging of ethanol-water surrogates demonstrated the ethanol concentration-radiodensity relationship is approximately linear. A concentration of 12% EC in ethanol created the largest distribution volume, more than eight-fold that of pure ethanol, ex vivo. In vivo, 12% EC-ethanol was superior to pure ethanol, yielding a distribution volume three-fold greater and an ablation zone six-fold greater than pure ethanol. Finally, a time course histological evaluation of the liver post-ablation with 12% EC-ethanol and pure ethanol revealed that while both induce coagulative necrosis and similar tissue responses at 1-4 weeks post-ablation, 12% EC-ethanol yielded a larger ablation zone. The current study demonstrates the suitability of CT imaging to determine distribution volume and concentration of ethanol in tissue. The distribution volume of EC-ethanol is nearly equivalent to the resultant necrotic volume and increases distribution and necrosis compared to pure ethanol.
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http://dx.doi.org/10.1038/s41598-021-99985-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8526742PMC
October 2021

β-Cyclodextrin-containing polymer treatment of cutaneous lupus and influenza improves outcomes.

Mol Ther 2021 Oct 8. Epub 2021 Oct 8.

Department of Surgery, Department of Pharmacology and Cancer Biology, Department of Neurosurgery, Department of Biomedical Engineering, Duke University, Durham, NC 27710, USA. Electronic address:

Nucleic acid (NA)-containing damage- and pathogen-associated molecular patterns (DAMPs and PAMPs, respectively) are implicated in numerous pathological conditions from infectious diseases to autoimmune disorders. Nucleic acid-binding polymers, including polyamidoamine (PAMAM) dendrimers, have demonstrated anti-inflammatory properties when administered to neutralize DAMPs/PAMPs. The PAMAM G3 variant has been shown to have beneficial effects in a cutaneous lupus erythematosus (CLE) murine model and improve survival of mice challenged with influenza. Unfortunately, the narrow therapeutic window of cationic PAMAM dendrimers makes their clinical development challenging. An alternative nucleic acid-binding polymer that has been evaluated in humans is a linear β-cyclodextrin-containing polymer (CDP). CDP's characteristics prompted us to evaluate its anti-inflammatory potential in CLE autoimmune and influenza infectious disease mouse models. We report that CDP effectively inhibits NA-containing DAMP-mediated activation of Toll-like receptors (TLRs) in cell culture, improves healing in lupus mice, and does not immunocompromise treated animals upon influenza infection but improves survival even when administered 3 days after infection. Finally, as anticipated, we observe limited toxicity in animals treated with CDP compared with PAMAM G3. Thus, CDP is a new anti-inflammatory agent that may be readily translated to the clinic to combat diseases associated with pathological NA-containing DAMPs/PAMPs.
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http://dx.doi.org/10.1016/j.ymthe.2021.10.003DOI Listing
October 2021

HER2 Isoforms Uniquely Program Intratumor Heterogeneity and Predetermine Breast Cancer Trajectories During the Occult Tumorigenic Phase.

Mol Cancer Res 2021 10 15;19(10):1699-1711. Epub 2021 Jun 15.

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina.

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (HER2), exon-16 null (HER2), and N-terminally truncated (HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although HER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, HER2 and HER2 induced rapid tumor development. HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for HER2 and HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. IMPLICATIONS: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment.
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http://dx.doi.org/10.1158/1541-7786.MCR-21-0215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507429PMC
October 2021

Poly(lactide-co-ε-caprolactone) scaffold promotes equivalent tissue integration and supports skin grafts compared to a predicate collagen scaffold.

Wound Repair Regen 2021 Nov 15;29(6):1035-1050. Epub 2021 Jun 15.

Division of Plastic and Reconstructive Surgery, Department of Surgery, School of Medicine, Duke University, Durham, North Carolina, USA.

Dermal scarring from motor vehicle accidents, severe burns, military blasts, etc. is a major problem affecting over 80 million people worldwide annually, many of whom suffer from debilitating hypertrophic scar contractures. These stiff, shrunken scars limit mobility, impact quality of life, and cost millions of dollars each year in surgical treatment and physical therapy. Current tissue engineered scaffolds have mechanical properties akin to unwounded skin, but these collagen-based scaffolds rapidly degrade over 2 months, premature to dampen contracture occurring 6-12 months after injury. This study demonstrates a tissue engineered scaffold can be manufactured from a slow-degrading viscoelastic copolymer, poly(ι-lactide-co-ε-caprolactone), with physical and mechanical characteristics to promote tissue ingrowth and support skin-grafts. Copolymers were synthesized via ring-opening polymerization. Solvent casting/particulate leaching was used to manufacture 3D porous scaffolds by mixing copolymers with particles in an organic solvent followed by casting into molds and subsequent particle leaching with water. Scaffolds characterized through SEM, micro-CT, and tensile testing confirmed the required thickness, pore size, porosity, modulus, and strength for promoting skin-graft bioincorporation and dampening fibrosis in vivo. Scaffolds were Oxygen Plasma Treatment and collagen coated to encourage cellular proliferation. Porosity ranging from 70% to 90% was investigated in a subcutaneous murine model and found to have no clinical effect on tissue ingrowth. A swine full-thickness skin wound model confirmed through histology and Computer Planimetry that scaffolds promote skin-graft survival, with or without collagen coating, with equal safety and efficacy as a commercially available tissue engineered scaffold. This study validates a scalable method to create poly(ι-lactide-co-ε-caprolactone) scaffolds with appropriate characteristics and confirms in mouse and swine wound models that the scaffolds are safe and effective at supporting skin-grafts. The results of this study have brought us closer towards developing an alternative technology that supports skin grafts with the potential to investigate long-term hypertrophic scar contractures.
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http://dx.doi.org/10.1111/wrr.12951DOI Listing
November 2021

Ex Vivo MR Histology and Cytometric Feature Mapping Connect Three-dimensional in Vivo MR Images to Two-dimensional Histopathologic Images of Murine Sarcomas.

Radiol Imaging Cancer 2021 05;3(3):e200103

From the Departments of Radiology (S.J.B., J.C., Y.Q., K.H., G.P.C., F.Z., C.T.B., G.A.J.), Radiation Oncology (Y.M.M., A.M.B., D.G.K.), and Pathology (J.I.E.), Duke University Medical Center, Center for In Vivo Microscopy, Bryan Research Building, 311 Research Dr, Durham, NC 27710.

Purpose To establish a platform for quantitative tissue-based interpretation of cytoarchitecture features from tumor MRI measurements. Materials and Methods In a pilot preclinical study, multicontrast in vivo MRI of murine soft-tissue sarcomas in 10 mice, followed by ex vivo MRI of fixed tissues (termed ), was performed. Paraffin-embedded limb cross-sections were stained with hematoxylin-eosin, digitized, and registered with MRI. Registration was assessed by using binarized tumor maps and Dice similarity coefficients (DSCs). Quantitative cytometric feature maps from histologic slides were derived by using nuclear segmentation and compared with registered MRI, including apparent diffusion coefficients and transverse relaxation times as affected by magnetic field heterogeneity (T2* maps). Cytometric features were compared with each MR image individually by using simple linear regression analysis to identify the features of interest, and the goodness of fit was assessed on the basis of values. Results Registration of MR images to histopathologic slide images resulted in mean DSCs of 0.912 for ex vivo MR histology and 0.881 for in vivo MRI. Triplicate repeats showed high registration repeatability (mean DSC, >0.9). Whole-slide nuclear segmentations were automated to detect nuclei on histopathologic slides (DSC = 0.8), and feature maps were generated for correlative analysis with MR images. Notable trends were observed between cell density and in vivo apparent diffusion coefficients (best line fit: = 0.96, < .001). Multiple cytoarchitectural features exhibited linear relationships with in vivo T2* maps, including nuclear circularity (best line fit: = 0.99, < .001) and variance in nuclear circularity (best line fit: = 0.98, < .001). Conclusion An infrastructure for registering and quantitatively comparing in vivo tumor MRI with traditional histologic analysis was successfully implemented in a preclinical pilot study of soft-tissue sarcomas. MRI, Pathology, Animal Studies, Tissue Characterization © RSNA, 2021.
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http://dx.doi.org/10.1148/rycan.2021200103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183263PMC
May 2021

Polymer-assisted intratumoral delivery of ethanol: Preclinical investigation of safety and efficacy in a murine breast cancer model.

PLoS One 2021 28;16(1):e0234535. Epub 2021 Jan 28.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.

Focal tumor ablation with ethanol could provide benefits in low-resource settings because of its low overall cost, minimal imaging technology requirements, and acceptable clinical outcomes. Unfortunately, ethanol ablation is not commonly utilized because of a lack of predictability of the ablation zone, caused by inefficient retention of ethanol at the injection site. To create a predictable zone of ablation, we have developed a polymer-assisted ablation method using ethyl cellulose (EC) mixed with ethanol. EC is ethanol-soluble and water-insoluble, allowing for EC-ethanol to be injected as a liquid and precipitate into a solid, occluding the leakage of ethanol upon contact with tissue. The aims of this study were to compare the 1) safety, 2) release kinetics, 3) spatial distribution, 4) necrotic volume, and 5) overall survival of EC-ethanol to conventional ethanol ablation in a murine breast tumor model. Non-target tissue damage was monitored through localized adverse events recording, ethanol release kinetics with Raman spectroscopy, injectate distribution with in vivo imaging, target-tissue necrosis with NADH-diaphorase staining, and overall survival by proxy of tumor growth. EC-ethanol exhibited decreased localized adverse events, a slowing of the release rate of ethanol, more compact injection zones, 5-fold increase in target-tissue necrosis, and longer overall survival rates compared to the same volume of pure ethanol. A single 150 μL dose of 6% EC-ethanol achieved a similar survival probability rates to six daily 50 μL doses of pure ethanol used to simulate a slow-release of ethanol over 6 days. Taken together, these results demonstrate that EC-ethanol is safer and more effective than ethanol alone for ablating tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234535PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843014PMC
September 2021

A Comparative Oncology Drug Discovery Pipeline to Identify and Validate New Treatments for Osteosarcoma.

Cancers (Basel) 2020 Nov 11;12(11). Epub 2020 Nov 11.

Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

Background: Osteosarcoma is a rare but aggressive bone cancer that occurs primarily in children. Like other rare cancers, treatment advances for osteosarcoma have stagnated, with little improvement in survival for the past several decades. Developing new treatments has been hampered by extensive genomic heterogeneity and limited access to patient samples to study the biology of this complex disease.

Methods: To overcome these barriers, we combined the power of comparative oncology with patient-derived models of cancer and high-throughput chemical screens in a cross-species drug discovery pipeline.

Results: Coupling in vitro high-throughput drug screens on low-passage and established cell lines with in vivo validation in patient-derived xenografts we identify the proteasome and CRM1 nuclear export pathways as therapeutic sensitivities in osteosarcoma, with dual inhibition of these pathways inducing synergistic cytotoxicity.

Conclusions: These collective efforts provide an experimental framework and set of new tools for osteosarcoma and other rare cancers to identify and study new therapeutic vulnerabilities.
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http://dx.doi.org/10.3390/cancers12113335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696249PMC
November 2020

Antibiotic eluting poly(ester urea) films for control of a model cardiac implantable electronic device infection.

Acta Biomater 2020 07 22;111:65-79. Epub 2020 May 22.

Department of Chemistry, Mechanical Engineering and Materials Science, Orthopaedic Surgery, and Biomedical Engineering, Duke University, Durham, NC, 27708, United States. Electronic address:

Cardiac implantable electronic device (CIED) infections acquired during or after surgical procedures are a major complication that are challenging to treat therapeutically, resulting in chronic and sometimes fatal infections. Localized delivery of antibiotics at the surgical site could be used to supplement traditional systemic administration as a preventative measure. Herein, we investigate a cefazolin-eluting l-valine poly(ester urea) (PEU) films as a model system for localized antibiotic delivery for CIEDs. Poly(1-VAL-8) PEU was used to fabricate a series of antibiotic-loaded films with varied loading concentrations (2%, 5%, 10% wt/wt) and thicknesses (40 µm, 80 µm, 140 µm). In vitro release measurements show thickness and loading concentration influence the amount and rate of cefazolin release. Group 10%-140 µm (load-thickness) showed 22.5% release of active pharmaceutical ingredient (API) in the first 24 h and 81.2% of cumulative percent release through day 14 and was found most effective in bacterial clearance in vitro. This group was also effective in clearing a bacterial infection in a model in vivo rat study while eliciting a limited inflammatory response. Our results suggest the feasibility of cefazolin-loaded PEU films as an effective sustained release matrix for localized delivery of antibiotics. SIGNIFICANCE STATEMENT: Implant-associated infections acquired during surgical procedures are a major complication that have proven a challenge to treat clinically, resulting in chronic and sometimes fatal infections. In this manuscript, we investigate an antibiotic-eluting L-valine poly(ester urea) (PEU) films as a model system for localized delivery of cefazolin. Significantly, we demonstrate a wide variation in temporal delivery and dosing within this family of PEUs and show that the delivery can be extended by varying the film thickness. The in vivo results show efficacy in an infected wound model and suggest antibiotic loaded PEU films function as an effective sustained release matrix for localized delivery of antibiotics across a number of clinical indications.
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http://dx.doi.org/10.1016/j.actbio.2020.04.025DOI Listing
July 2020

Comparisons of Infant and Adult Mice Reveal Age Effects for Liver Depot Gene Therapy in Pompe Disease.

Mol Ther Methods Clin Dev 2020 Jun 6;17:133-142. Epub 2019 Dec 6.

Division of Medical Genetics, Duke University School of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

Pompe disease is caused by the deficiency of lysosomal acid α-glucosidase (GAA). It is expected that gene therapy to replace GAA with adeno-associated virus (AAV) vectors will be less effective early in life because of the rapid loss of vector genomes. AAV2/8-LSPhGAA (3 × 10 vector genomes [vg]/mouse) was administered to infant (2-week-old) or adult (2-month-old) GAA knockout mice. AAV vector transduction in adult mice significantly corrected GAA deficiency in the heart (p < 0.0001), diaphragm (p < 0.01), and quadriceps (p < 0.001) for >50 weeks. However, in infant mice, the same treatment only partially corrected GAA deficiency in the heart (p < 0.05), diaphragm (p < 0.05), and quadriceps (p < 0.05). The clearance of glycogen was much more efficient in adult mice compared with infant mice. Improved wire hang test latency was observed for treated adults (p < 0.05), but not for infant mice. Abnormal ventilation was corrected in both infant and adult mice. Vector-treated female mice demonstrated functional improvement, despite a lower degree of biochemical correction compared with male mice. The relative vector dose for infants was approximately 3-fold higher than adults, when normalized to body weight at the time of vector administration. Given these data, the dose requirement to achieve similar efficacy will be higher for the treatment of young patients.
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http://dx.doi.org/10.1016/j.omtm.2019.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938806PMC
June 2020

Salmeterol with Liver Depot Gene Therapy Enhances the Skeletal Muscle Response in Murine Pompe Disease.

Hum Gene Ther 2019 07 5;30(7):855-864. Epub 2019 Apr 5.

1Division of Medical Genetics, Department of Pediatrics, Duke University Medical School, Durham, North Carolina.

Gene therapy for Pompe disease with adeno-associated virus (AAV) vectors has advanced into early phase clinical trials; however, the paucity of cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle, where it is needed to take up acid α-glucosidase (GAA), has impeded the efficacy of Pompe disease gene therapy. Long-acting selective β2 receptor agonists previously enhanced the CI-MPR expression in muscle. In this study we have evaluated the selective β2 agonist salmeterol in GAA knockout mice in combination with an AAV vector expressing human GAA specifically in the liver. Quadriceps glycogen content was significantly decreased by administration of the AAV vector with salmeterol, in comparison with the AAV vector alone ( < 0.01). Importantly, glycogen content of the quadriceps was reduced to its lowest level by the combination of AAV vector and salmeterol administration. Rotarod testing revealed significant improvement following treatment, in comparison with untreated mice, and salmeterol improved wirehang performance. Salmeterol treatment decreased abnormalities of autophagy in the quadriceps, as shown be lower LC3 and p62. Vector administration reduced the abnormal vacuolization and accumulation of nuclei in skeletal muscle. Thus, salmeterol could be further developed as adjunctive therapy to improve the efficacy of liver depot gene therapy for Pompe disease.
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http://dx.doi.org/10.1089/hum.2018.197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648189PMC
July 2019

Author Correction: Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors.

Nat Med 2019 Mar;25(3):529

Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA.

In the version of this article originally published, the figure callout in this sentence was incorrect: "Furthermore, in S1P1-KI mice themselves, whereas PD-1 blockade was ineffectual as monotherapy, the effects of 4-1BB agonism and checkpoint blockade proved additive, with the combination prolonging median survival and producing a 50% long-term survival rate (Fig. 6f)." The callout should have been to Supplementary Fig. 6b. The error has been corrected in the PDF and HTML versions of the article.
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http://dx.doi.org/10.1038/s41591-019-0355-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825406PMC
March 2019

Pathology Study Design, Conduct, and Reporting to Achieve Rigor and Reproducibility in Translational Research Using Animal Models.

ILAR J 2018 12;59(1):4-12

Department of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut.

In translational research, animal models are an important tool to aid in decision-making when taking potential therapies into human clinical trials. Recently, there have been a number of papers that have suggested limited concordance of preclinical animal experiments with subsequent human clinical experience. Assessments of preclinical animal studies have led to concerns about the reproducibility of data and have highlighted the need for an emphasis on rigor and quality in the planning, conduct, analysis, and reporting of such studies. The incorporation of a wider role for the comparative pathologist using pathology best practices in the planning and conduct of animal model-based research is one way to increase the quality and reproducibility of data. The use of optimal design and planning of tissue collection, incorporation of pathology methods into written protocols, conduct of pathology procedures using accepted best practices, and the use of optimal pathology analysis and reporting methods enhance the quality of the data acquired from many types of preclinical animal models and studies. Many of these pathology practices are well established in the discipline of toxicologic pathology and have a proven and useful track record in enhancing the data from animal-based studies used in safety assessment of human therapeutics. Some of this experience can be adopted by the wider community of preclinical investigators to increase the reproducibility of animal study data.
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http://dx.doi.org/10.1093/ilar/ily020DOI Listing
December 2018

Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors.

Nat Med 2018 09 13;24(9):1459-1468. Epub 2018 Aug 13.

Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA.

T cell dysfunction contributes to tumor immune escape in patients with cancer and is particularly severe amidst glioblastoma (GBM). Among other defects, T cell lymphopenia is characteristic, yet often attributed to treatment. We reveal that even treatment-naïve subjects and mice with GBM can harbor AIDS-level CD4 counts, as well as contracted, T cell-deficient lymphoid organs. Missing naïve T cells are instead found sequestered in large numbers in the bone marrow. This phenomenon characterizes not only GBM but a variety of other cancers, although only when tumors are introduced into the intracranial compartment. T cell sequestration is accompanied by tumor-imposed loss of S1P1 from the T cell surface and is reversible upon precluding S1P1 internalization. In murine models of GBM, hindering S1P1 internalization and reversing sequestration licenses T cell-activating therapies that were previously ineffective. Sequestration of T cells in bone marrow is therefore a tumor-adaptive mode of T cell dysfunction, whose reversal may constitute a promising immunotherapeutic adjunct.
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http://dx.doi.org/10.1038/s41591-018-0135-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129206PMC
September 2018

Long-term complications of glycogen storage disease type Ia in the canine model treated with gene replacement therapy.

J Inherit Metab Dis 2018 11 24;41(6):965-976. Epub 2018 Jul 24.

Division of Medical Genetics, Duke University Medical Center (DUMC), Box 103856, Durham, NC, 27710, USA.

Background: Glycogen storage disease type Ia (GSD Ia) in dogs closely resembles human GSD Ia. Untreated patients with GSD Ia develop complications associated with glucose-6-phosphatase (G6Pase) deficiency. Survival of human patients on intensive nutritional management has improved; however, long-term complications persist including renal failure, nephrolithiasis, hepatocellular adenomas (HCA), and a high risk for hepatocellular carcinoma (HCC). Affected dogs fail to thrive with dietary therapy alone. Treatment with gene replacement therapy using adeno-associated viral vectors (AAV) expressing G6Pase has greatly prolonged life and prevented hypoglycemia in affected dogs. However, long-term complications have not been described to date.

Methods: Five GSD Ia-affected dogs treated with AAV-G6Pase were evaluated. Dogs were euthanized due to reaching humane endpoints related to liver and/or kidney involvement, at 4 to 8 years of life. Necropsies were performed and tissues were analyzed.

Results: Four dogs had liver tumors consistent with HCA and HCC. Three dogs developed renal failure, but all dogs exhibited progressive kidney disease histologically. Urolithiasis was detected in two dogs; uroliths were composed of calcium oxalate and calcium phosphate. One affected and one carrier dog had polycystic ovarian disease. Bone mineral density was not significantly affected.

Conclusions: Here, we show that the canine GSD Ia model demonstrates similar long-term complications as GSD Ia patients in spite of gene replacement therapy. Further development of gene therapy is needed to develop a more effective treatment to prevent long-term complications of GSD Ia.
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http://dx.doi.org/10.1007/s10545-018-0223-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328337PMC
November 2018

Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader-Willi syndrome.

Nat Med 2017 Feb 26;23(2):213-222. Epub 2016 Dec 26.

Department of Pediatrics, School of Medicine, Duke University, Durham, North Carolina, USA.

Prader-Willi syndrome (PWS) is an imprinting disorder caused by a deficiency of paternally expressed gene(s) in the 15q11-q13 chromosomal region. The regulation of imprinted gene expression in this region is coordinated by an imprinting center (PWS-IC). In individuals with PWS, genes responsible for PWS on the maternal chromosome are present, but repressed epigenetically, which provides an opportunity for the use of epigenetic therapy to restore expression from the maternal copies of PWS-associated genes. Through a high-content screen (HCS) of >9,000 small molecules, we discovered that UNC0638 and UNC0642-two selective inhibitors of euchromatic histone lysine N-methyltransferase-2 (EHMT2, also known as G9a)-activated the maternal (m) copy of candidate genes underlying PWS, including the SnoRNA cluster SNORD116, in cells from humans with PWS and also from a mouse model of PWS carrying a paternal (p) deletion from small nuclear ribonucleoprotein N (Snrpn (S)) to ubiquitin protein ligase E3A (Ube3a (U)) (mouse model referred to hereafter as m/p). Both UNC0642 and UNC0638 caused a selective reduction of the dimethylation of histone H3 lysine 9 (H3K9me2) at PWS-IC, without changing DNA methylation, when analyzed by bisulfite genomic sequencing. This indicates that histone modification is essential for the imprinting of candidate genes underlying PWS. UNC0642 displayed therapeutic effects in the PWS mouse model by improving the survival and the growth of m/p newborn pups. This study provides the first proof of principle for an epigenetics-based therapy for PWS.
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http://dx.doi.org/10.1038/nm.4257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589073PMC
February 2017

HIV-1 Envelope Mimicry of Host Enzyme Kynureninase Does Not Disrupt Tryptophan Metabolism.

J Immunol 2016 12 14;197(12):4663-4673. Epub 2016 Nov 14.

Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710;

The HIV-1 envelope protein (Env) has evolved to subvert the host immune system, hindering viral control by the host. The tryptophan metabolic enzyme kynureninase (KYNU) is mimicked by a portion of the HIV Env gp41 membrane proximal region (MPER) and is cross-reactive with the HIV broadly neutralizing Ab (bnAb) 2F5. Molecular mimicry of host proteins by pathogens can lead to autoimmune disease. In this article, we demonstrate that neither the 2F5 bnAb nor HIV MPER-KYNU cross-reactive Abs elicited by immunization with an MPER peptide-liposome vaccine in 2F5 bnAb VDJ and VJ knock-in mice and rhesus macaques modified KYNU activity or disrupted tissue tryptophan metabolism. Thus, molecular mimicry by HIV-1 Env that promotes the evasion of host anti-HIV-1 Ab responses can be directed toward nonfunctional host protein epitopes that do not impair host protein function. Therefore, the 2F5 HIV Env gp41 region is a key and safe target for HIV-1 vaccine development.
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http://dx.doi.org/10.4049/jimmunol.1601484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136304PMC
December 2016

Recommendations for Addressing Harm-Benefit Analysis and Implementation in Ethical Evaluation - Report from the AALAS-FELASA Working Group on Harm-Benefit Analysis - Part 2.

Lab Anim 2016 Jun;50(1 Suppl):21-42

University of Bergen, Department of Clinical Medicine, Bergen, Norway.

International regulations and guidelines strongly suggest that the use of animal models in scientific research should be initiated only after the authority responsible for the review of animal studies has concluded a well-thought-out harm-benefit analysis (HBA) and deemed the project to be appropriate. The AALAS-FELASA working group on HBA has performed a literature review and based on this review, proposed a method for HBA. Examples of the working group's approach are included in this report.
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http://dx.doi.org/10.1177/0023677216642397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815834PMC
June 2016

Current concepts of Harm-Benefit Analysis of Animal Experiments - Report from the AALAS-FELASA Working Group on Harm-Benefit Analysis - Part 1.

Lab Anim 2016 Jun;50(1 Suppl):1-20

Chief, Comparative Medicine Branch, NIEHS/NIH, Research Triangle Park, NC, USA.

International regulations and guidelines strongly suggest that the use of animal models in scientific research should be initiated only after the authority responsible for the review of animal studies has concluded a well-thought-out harm-benefit analysis (HBA) and deemed the project to be appropriate. Although the process for conducting HBAs may not be new, the relevant factors and algorithms used in conducting them during the review process are deemed to be poorly defined or lacking by committees in many institutions. This paper presents the current concept of HBAs based on a literature review. References on cost or risk benefit from clinical trials and other industries are also included. Several approaches to HBA have been discovered including algorithms, graphic presentations and generic processes. The aim of this study is to better aid and harmonize understanding of the concepts of 'harm', 'benefit' and 'harm-benefit analysis'.
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http://dx.doi.org/10.1177/0023677216642398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815836PMC
June 2016

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

The future of preclinical animal models in pharmaceutical discovery and development: a need to bring in cerebro to the in vivo discussions.

Toxicol Pathol 2015 Jan 27;43(1):70-7. Epub 2014 Oct 27.

GlaxoSmithKline, Research Triangle Park, North Carolina, USA

Animal models have provided an important tool to help make the decision to take potential therapies from preclinical studies to humans. In the past several years, the strong reliance of the pharmaceutical discovery and development process on the use of animal models has come under increasing scrutiny for ethical and scientific reasons. Several prominent and widely publicized articles have reported limited concordance of animal experiments with subsequent human clinical trials. Recent assessments of the quality of animal studies have suggested that this translational failure may be due in part to shortcomings in the planning, conduct, and reporting of in vivo studies. This article will emphasize methods to assure best practice rigor in animal study methods and reporting. It will introduce the so-called scientific 3Rs of relevance, robustness, and reproducibility to the in vivo study approach and will review important new trends in the animal research and pharmaceutical discovery and development communities.
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http://dx.doi.org/10.1177/0192623314555162DOI Listing
January 2015

Environmental estrogens differentially engage the histone methyltransferase EZH2 to increase risk of uterine tumorigenesis.

Mol Cancer Res 2012 Apr;10(4):546-57

Science Park Research Division, Department of Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas, USA.

Environmental exposures during sensitive windows of development can reprogram normal physiologic responses and alter disease susceptibility later in life in a process known as developmental reprogramming. For example, exposure to the xenoestrogen diethylstilbestrol during reproductive tract development can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyperresponsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein, a soy phytoestrogen, and the plasticizer bisphenol A, differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. Whereas both genistein and bisphenol A induce genomic estrogen receptor (ER) signaling in the developing uterus, only genistein induced phosphoinositide 3-kinase (PI3K)/AKT nongenomic ER signaling to the histone methyltransferase enhancer of zeste homolog 2 (EZH2). As a result, this pregenomic signaling phosphorylates and represses EZH2 and reduces levels of H3K27me3 repressive mark in chromatin. Furthermore, only genistein caused estrogen-responsive genes in the adult myometrium to become hyperresponsive to hormone; estrogen-responsive genes were repressed in bisphenol A-exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by genistein promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, whereas bisphenol A did not. These data show that environmental estrogens have distinct nongenomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis.
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http://dx.doi.org/10.1158/1541-7786.MCR-11-0605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879949PMC
April 2012

Non-neoplastic and neoplastic pleural endpoints following fiber exposure.

J Toxicol Environ Health B Crit Rev 2011 ;14(1-4):153-78

University of California, San Francisco, San Francisco, California, USA.

Exposure to asbestos fibers is associated with non-neoplastic pleural diseases including plaques, fibrosis, and benign effusions, as well as with diffuse malignant pleural mesothelioma. Translocation and retention of fibers are fundamental processes in understanding the interactions between the dose and dimensions of fibers retained at this anatomic site and the subsequent pathological reactions. The initial interaction of fibers with target cells in the pleura has been studied in cellular models in vitro and in experimental studies in vivo. The proposed biological mechanisms responsible for non-neoplastic and neoplastic pleural diseases and the physical and chemical properties of asbestos fibers relevant to these mechanisms are critically reviewed. Understanding mechanisms of asbestos fiber toxicity may help us anticipate the problems from future exposures both to asbestos and to novel fibrous materials such as nanotubes. Gaps in our understanding have been outlined as guides for future research.
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http://dx.doi.org/10.1080/10937404.2011.556049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3118521PMC
July 2011

Pleural effects of indium phosphide in B6C3F1 mice: nonfibrous particulate induced pleural fibrosis.

Exp Lung Res 2009 Dec;35(10):858-82

Respiratory Toxicology, Laboratory of Molecular Toxicology, Environmental Toxicology Program/National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.

The mechanism(s) by which chronic inhalation of indium phosphide (InP) particles causes pleural fibrosis is not known. Few studies of InP pleural toxicity have been conducted because of the challenges in conducting particulate inhalation exposures, and because the pleural lesions developed slowly over the 2-year inhalation study. The authors investigated whether InP (1 mg/kg) administered by a single oropharyngeal aspiration would cause pleural fibrosis in male B6C3F1 mice. By 28 days after treatment, protein and lactate dehydrogenase (LDH) were significantly increased in bronchoalveolar lavage fluid (BALF), but were unchanged in pleural lavage fluid (PLF). A pronounced pleural effusion characterized by significant increases in cytokines and a 3.7-fold increase in cell number was detected 28 days after InP treatment. Aspiration of soluble InCl(3) caused a similar delayed pleural effusion; however, other soluble metals, insoluble particles, and fibers did not. The effusion caused by InP was accompanied by areas of pleural thickening and inflammation at day 28, and by pleural fibrosis at day 98. Aspiration of InP produced pleural fibrosis that was histologically similar to lesions caused by chronic inhalation exposure, and in a shorter time period. This oropharyngeal aspiration model was used to provide an initial characterization of the progression of pleural lesions caused by InP.
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http://dx.doi.org/10.3109/01902140902980961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2928993PMC
December 2009

Inhaled carbon nanotubes reach the subpleural tissue in mice.

Nat Nanotechnol 2009 Nov 25;4(11):747-51. Epub 2009 Oct 25.

Department of Environmental and Molecular Toxicology, College of Agricultural and Life Sciences, North Carolina State University, Raleigh, NC 27695, USA.

Carbon nanotubes are shaped like fibres and can stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice, raising concerns that inhaled nanotubes may cause pleural fibrosis and/or mesothelioma. Here, we show that multiwalled carbon nanotubes reach the subpleura in mice after a single inhalation exposure of 30 mg m(-3) for 6 h. Nanotubes were embedded in the subpleural wall and within subpleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Subpleural fibrosis unique to this form of nanotubes increased after 2 and 6 weeks following inhalation. None of these effects was seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg m(-3)). This work suggests that minimizing inhalation of nanotubes during handling is prudent until further long-term assessments are conducted.
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http://dx.doi.org/10.1038/nnano.2009.305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783215PMC
November 2009

Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats.

Clin Cancer Res 2007 May;13(10):3087-99

GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania, USA.

Purpose: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions.

Experimental Design: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency.

Results: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals.

Conclusion: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.
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http://dx.doi.org/10.1158/1078-0432.CCR-06-1811DOI Listing
May 2007

Preparation of animals for use in the laboratory: issues and challenges for the Institutional Animal Care and Use Committee (IACUC).

ILAR J 2006 ;47(4):370-5

Department of Comparative Medicine, Michale E. Keeling Center for Comparative Medicine and Research of The University of Texas M. D. Anderson Cancer Center, Bastrop, TX, USA.

Preparation of animals is important for optimization of animal welfare as well as to minimize interanimal variation, thereby strengthening the quality of data for in vivo studies. These issues are important in the work of institutional animal care and use committees (IACUCs), but they pose many challenges as well. This article provides IACUC members with a resource for use in determining whether and how preparation of animals for research affects the IACUC's responsibilities. The topics addressed are intended to serve as a starting point for consideration and discussion. Questions related to subject selection and acclimation of subjects to experimental housing and procedures are emphasized and should provide IACUC members with a framework for discussion of relevant questions. Guidelines are provided for promoting the acclimation of a number of species to experimental settings. Additional, potentially controversial points are also raised, including the effects on longitudinal data sets of changing subject preparation procedures. The roles of the IACUC in the research endeavor are multifaceted and continuously evolving. As empirical data are produced that affect additional aspects of animal care and use, it is important for these committees to be able to evaluate and, when appropriate, stimulate the implementation of improved procedures and strategies.
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http://dx.doi.org/10.1093/ilar.47.4.370DOI Listing
October 2006

The art and science of introducing animals to the research environment.

ILAR J 2006 ;47(4):281-2

Department of Comparative Biology & Medicine, GlaxoSmithKline Research & Development, Research Triangle Park, NC, USA.

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http://dx.doi.org/10.1093/ilar.47.4.281DOI Listing
October 2006

Analysis of renal cell transformation following exposure to trichloroethene in vivo and its metabolite S-(dichlorovinyl)-L-cysteine in vitro.

Toxicology 2006 Jul 28;224(1-2):108-18. Epub 2006 Apr 28.

Department of Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany.

Trichloroethene (TCE) is classified as a potential human carcinogen although there is a significant debate regarding the mechanism of TCE induced renal tumor formation. This controversy stems in part from the extremely high doses of TCE required to induce renal tumors and the potential contribution of the associated nephrotoxicity to tumorigenesis. We have used Eker rats, which are uniquely susceptible to renal carcinogens, to determine if exposures to TCE in vivo or exposure to its metabolite S-(dichlorovinyl)-L-cysteine (DCVC) in vitro can transform kidney epithelial cells in the absence of cytotoxicity. Treatment with TCE (0, 100, 250, 500, 1000 mg/kg bw by gavage, 5 days a week) for 13 weeks resulted in a significant increase in cell proliferation in kidney tubule cells, but did not enhance formation of preneoplastic lesions or tumor incidence in Eker rat kidneys as compared to controls. In vitro, concentrations of DCVC, which reduced cell survival to 50%, were able to transform rat kidney epithelial cells. However, no carcinogen-specific mutations were identified in the VHL or Tsc-2 tumor suppressor genes in the transformants. Taken together, the inability of TCE to enhance formation of preneoplastic changes or neoplasia and the absence of carcinogen-specific alteration of genes accepted to be critical for renal tumor development suggest that TCE mediated carcinogenicity may occur secondary to continuous toxic injury and sustained regenerative cell proliferation.
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http://dx.doi.org/10.1016/j.tox.2006.04.036DOI Listing
July 2006

What's your diagnosis? Lethargy and hind limb paralysis in a day-23 timed pregnant rat.

Lab Anim (NY) 2006 Feb;35(2):19-20

CIIT Centers for Health Research, 6 Davis Dr., PO Box 12137, Research Triangle Park, NC 27709, USA.

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http://dx.doi.org/10.1038/laban0206-19DOI Listing
February 2006

Laboratory animal science issues in the design and conduct of studies with endocrine-active compounds.

ILAR J 2004 ;45(4):417-24

GlaxoSmithKline Pharmaceutical Research and Development, Research Triangle Park, NC, USA.

The use of rodent models for research and testing on endocrine-active compounds necessitates an awareness of a number of laboratory animal science issues to standardize bioassay methods and facilitate reproducibility of results between laboratories. These issues are not unique to endocrine research but are particularly important in this field due to the complexities and interdependencies of the endocrine system, coupled with the inherently sensitive and variable nature of physiological endpoints. Standardization of animal models and the control of animal environments depend on the establishment of strong scientific partnerships between research investigators and laboratory animal scientists. Laboratory animal care and use programs are becoming increasingly complex and are constantly changing, fueled in part by technological advances, changes in regulations concerning animal care and use, and economic pressures. Since the early 1980s, many institutions have moved to centralization of animal facility operations concomitant with numerous changes in housing systems, barrier concepts, equipment, and engineering controls of the macro- and microenvironment. These and other changes can have an impact on animals and the conduct of endocrine experiments. Despite the potential impact of animal care and use procedures on research endpoints, many investigators are surprisingly naive to the animal facility conditions that can affect in vivo studies. Several key animal care and use issues that are important to consider in endocrine experiments with rodent models are described.
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http://dx.doi.org/10.1093/ilar.45.4.417DOI Listing
July 2005
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