Publications by authors named "David K Meyerholz"

202 Publications

V-type ATPase Mediates Airway Surface Liquid Acidification in Pig Small Airway Epithelial Cells.

Am J Respir Cell Mol Biol 2021 Mar 31. Epub 2021 Mar 31.

The University of Iowa, 4083, Internal Medicine, Iowa City, Iowa, United States;

In a new born pig cystic fibrosis (CF) model, the ability of gland-containing airways to fight infection was affected by at least two major host-defense defects: impaired mucociliary transport and a lower airway-surface liquid (ASL) pH. In the gland-containing airways, ASL pH is balanced by CFTR and ATP12A, which respectively control HCO3- transport and proton secretion. We found that, although porcine small airway tissue expressed little ATP12A, the ASL of epithelial cultures from CF distal small airways (diameter <200 μm) were nevertheless more acidic (compared to non-CF). Therefore, we hypothesized that gland-containing airways vs. small airways control acidification using distinct mechanisms. Our microarray data suggested that small airway epithelia mediate proton secretion via ATP6V0D2, an isoform of the V0d subunit of the H+-translocating plasma membrane V-type ATPase. Immunofluorescence of small airways verified the expression of the V0d2 subunit isoform at the apical surface of Muc5B+ secretory cells, but not ciliated cells. Inhibiting the V-type ATPase with bafilomycin A1 elevated the ASL pH of small airway cultures, in the presence or absence of HCO3-, and decreased ASL viscosity. These data suggest that, unlike large airways, which are acidified by ATP12A activity, small airways are acidified by V-type ATPase, thus identifying V-type ATPase as a novel therapeutic target for small airways diseases.
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http://dx.doi.org/10.1165/rcmb.2020-0349OCDOI Listing
March 2021

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

Challenges and Opportunities for the Veterinary Pathologist in Biomedical Research.

Vet Pathol 2021 Mar 17;58(2):258-265. Epub 2020 Dec 17.

Albert Einstein College of Medicine, Bronx, NY, USA.

Animal models have critical roles in biomedical research in promoting understanding of human disease and facilitating development of new therapies and diagnostic techniques to improve human and animal health. In the study of myriad human conditions, each model requires in-depth characterization of its assets and limitations in order for it to be used to greatest advantage. Veterinary pathology expertise is critical in understanding the relevance and translational validity of animal models to conditions under study, assessing morbidity and mortality, and validating outcomes as relevant or not to the study interventions. Clear communication with investigators and education of research personnel on the use and interpretation of pathology endpoints in animal models are critical to the success of any research program. The veterinary pathologist is underutilized in biomedical research due to many factors including misconceptions about high fiscal costs, lack of perceived value, limited recognition of their expertise, and the generally low number of veterinary pathologists currently employed in biomedical research. As members of the multidisciplinary research team, veterinary pathologists have an important role to educate scientists, ensure accurate interpretation of pathology data, maximize rigor, and ensure reproducibility to provide the most reliable data for animal models in biomedical research.
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http://dx.doi.org/10.1177/0300985820974005DOI Listing
March 2021

Severe Acute Respiratory Syndrome Coronavirus 2-Induced Immune Activation and Death of Monocyte-Derived Human Macrophages and Dendritic Cells.

J Infect Dis 2021 03;223(5):785-795

Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

Studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients and experimentally infected animals indicate a critical role for augmented expression of proinflammatory chemokines and cytokines in severe disease. Here, we demonstrate that SARS-CoV-2 infection of human monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells was abortive, but induced the production of multiple antiviral and proinflammatory cytokines (interferon-α, interferon-β, tumor necrosis factor, and interleukins 1β, 6, and 10) and a chemokine (CXCL10). Despite the lack of efficient replication in MDMs, SARS-CoV-2 induced profound interferon-mediated cell death of host cells. Macrophage activation and death were not enhanced by exposure to low levels of convalescent plasma, suggesting that antibody-dependent enhancement of infection does not contribute to cell death. Together, these results indicate that infection of macrophages and dendritic cells potentially plays a major role in coronavirus disease 2019 pathogenesis, even in the absence of productive infection.
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http://dx.doi.org/10.1093/infdis/jiaa753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799009PMC
March 2021

Does common cold coronavirus infection protect against severe SARS-CoV-2 disease?

J Clin Invest 2021 01;131(1)

Department of Microbiology and Immunology, and.

The coronavirus disease 2019 (COVID-19) pandemic continues to cause morbidity and mortality. Since SARS coronavirus 2 (SARS-CoV-2) was identified as the cause for COVID-19, some have questioned whether exposure to seasonal common cold coronaviruses (CCCs) could provide tangible protection against SARS-CoV-2 infection or disease. In this issue of the JCI, Sagar et al. examined SARS-CoV-2 infections and outcomes of patients who had previously tested positive or negative for CCC infection (CCC+ or CCC-) by a comprehensive respiratory panel using PCR. No differences were seen between groups in terms of susceptibility to SARS-CoV-2 infection. However, hospitalized patients with a documented history of CCC infection had lower rates of intensive care unit (ICU) admissions and higher rates of survival than hospitalized CCC- patients. While these findings are associative and not causative, they highlight evidence suggesting that previous CCC infection may influence the disease course of SARS-CoV-2 infection.
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http://dx.doi.org/10.1172/JCI144807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773392PMC
January 2021

COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice.

Nature 2021 01 9;589(7843):603-607. Epub 2020 Nov 9.

Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is associated with substantial morbidity and mortality. Although much has been learned in the first few months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation, and many patients with anosmia show no or only minor respiratory symptoms. Studies in animals infected experimentally with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, provide opportunities to study aspects of the disease that are not easily investigated in human patients. Although the severity of COVID-19 ranges from asymptomatic to lethal, most experimental infections provide insights into mild disease. Here, using K18-hACE2 transgenic mice that were originally developed for SARS studies, we show that infection with SARS-CoV-2 causes severe disease in the lung and, in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Furthermore, we show that infusion of convalescent plasma from a recovered patient with COVID-19 protected against lethal disease. Mice developed anosmia at early time points after infection. Notably, although pre-treatment with convalescent plasma prevented most signs of clinical disease, it did not prevent anosmia. Thus, K18-hACE2 mice provide a useful model for studying the pathological basis of both mild and lethal COVID-19 and for assessing therapeutic interventions.
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http://dx.doi.org/10.1038/s41586-020-2943-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855185PMC
January 2021

Nonviral Gene Delivery Embedded in Biomimetically Mineralized Matrices for Bone Tissue Engineering.

Tissue Eng Part A 2020 Nov 26. Epub 2020 Nov 26.

Department of Orthodontics, University of Iowa College of Dentistry and Dental Clinics, Iowa City, Iowa, USA.

Research in bone tissue engineering aims to design materials that are effective at generating bone without causing significant side effects. The osteogenic potential of combining matrices and protein growth factors has been well documented, however, improvements are necessary to achieve optimal therapeutic benefits upon clinical translation. In this article, rat calvarial defects were treated with gene-activated matrices (GAMs). The GAMs used were collagen sponges mineralized with a simulated body fluid (SBF) containing a nonviral gene delivery system. Both and studies were performed to determine the optimal mode of gene delivery. After 6 weeks, the defects were extracted to assess bone formation and tissue quality through histological and microcomputed tomography analyses. The optimal GAM consisted of a collagen sponge with polyethylenimine plasmid DNA (PEI-pDNA) complexes embedded in a calcium phosphate coating produced by SBF, which increased total bone formation by 39% compared with 19% for control samples. A follow-up study was performed to optimize the ratio of growth factors included in the GAM. The optimal ratio for supporting bone formation after 6 weeks of implantation was five parts of pBMP-2 to three parts pFGF-2. These studies demonstrated that collagen matrices biomimetically mineralized and activated with plasmids encoding fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) can optimally improve bone regeneration outcomes. Impact statement Bone tissue engineering has explored both nonviral gene delivery and the concept of biomimetic mineralization. In this study, we combined these two concepts to further enhance bone regeneration outcomes. We demonstrated that embedding polyethylenimine (PEI)-based gene delivery within a mineral layer formed from simulated body fluid (SBF) immersion can increase bone formation rates. We also demonstrated that the ratio of growth factors utilized for matrix fabrication can impact the amount of bone formed in the defect site. This research highlights a combined approach using SBF and nonviral gene delivery both and and prepares the way for future optimization of synthetic gene activated matrices.
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http://dx.doi.org/10.1089/ten.TEA.2020.0206DOI Listing
November 2020

Lack of airway submucosal glands impairs respiratory host defenses.

Elife 2020 10 7;9. Epub 2020 Oct 7.

Department of Internal Medicine and Pappajohn Biomedical Institute Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, United States.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (), which initiates SMG development. pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in pigs. Consequently, pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.
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http://dx.doi.org/10.7554/eLife.59653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541087PMC
October 2020

Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract.

EBioMedicine 2020 Oct 21;60:102976. Epub 2020 Sep 21.

Departments of Pathology, University of Iowa College of Medicine, University of Iowa, Iowa City, IA, USA. Electronic address:

Background: Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors.

Materials And Methods: We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19.

Findings: Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19.

Interpretation: These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19.
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http://dx.doi.org/10.1016/j.ebiom.2020.102976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505653PMC
October 2020

Toxicity assessment of metal oxide nanomaterials using screening and murine acute inhalation studies.

NanoImpact 2020 Apr 20;18. Epub 2020 Feb 20.

Department of Occupational and Environmental Health, University of Iowa, Iowa City, IA 52246, USA.

Characterizations and toxicity screening were performed on metal oxide engineered nanomaterials (ENMs) independently comprising ZnO, CuO, CeO, FeO, WO, VO, TiO, AlO and MgO. Nanomaterials that exhibited the highest toxicity responses in the screening assays (ZnO, CuO, and VO) and the lesser explored material WO were tested for acute pulmonary toxicity . Female and male mice (C57Bl/6J) were exposed to aerosolized metal oxide ENMs in a nose-only exposure system and toxicity outcomes (biomarkers of cytotoxicity, immunotoxicity, inflammation, and lung histopathology) at 4 and 24 h after the start of exposure were assessed. The studies were performed as part of the NIEHS Nanomaterials Health Implications Research consortium with the purpose of investigating the effects of ENMs on various biological systems. ENMs were supplied by the Engineered Nanomaterials Resource and Coordination Core. Among the ENMs studied, the highest toxicity was observed for CuO and ZnO NPs in both and acute models. Compared to sham-exposed controls, there was a significant increase in bronchoalveolar lavage neutrophils and proinflammatory cytokines and a loss of macrophage viability at both 4 h and 24 h for ZnO and CuO but not seen for VO or WO. These effects were observed in both female and male mice. The cell viability performed after exposure to ENMs and assessment of lung inflammation after acute inhalation exposure were shown to be sensitive endpoints to predict ENM acute toxicity.
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http://dx.doi.org/10.1016/j.impact.2020.100214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504913PMC
April 2020

Microglia depletion exacerbates demyelination and impairs remyelination in a neurotropic coronavirus infection.

Proc Natl Acad Sci U S A 2020 09 14;117(39):24464-24474. Epub 2020 Sep 14.

Interdisciplinary Program in Immunology, University of Iowa, Iowa City, IA 52242;

Microglia are considered both pathogenic and protective during recovery from demyelination, but their precise role remains ill defined. Here, using an inhibitor of colony stimulating factor 1 receptor (CSF1R), PLX5622, and mice infected with a neurotropic coronavirus (mouse hepatitis virus [MHV], strain JHMV), we show that depletion of microglia during the time of JHMV clearance resulted in impaired myelin repair and prolonged clinical disease without affecting the kinetics of virus clearance. Microglia were required only during the early stages of remyelination. Notably, large deposits of extracellular vesiculated myelin and cellular debris were detected in the spinal cords of PLX5622-treated and not control mice, which correlated with decreased numbers of oligodendrocytes in demyelinating lesions in drug-treated mice. Furthermore, gene expression analyses demonstrated differential expression of genes involved in myelin debris clearance, lipid and cholesterol recycling, and promotion of oligodendrocyte function. The results also demonstrate that microglial functions affected by depletion could not be compensated by infiltrating macrophages. Together, these results demonstrate that microglia play key roles in debris clearance and in the initiation of remyelination following infection with a neurotropic coronavirus but are not necessary during later stages of remyelination.
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http://dx.doi.org/10.1073/pnas.2007814117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533697PMC
September 2020

K18-hACE2 Mice for Studies of COVID-19 Treatments and Pathogenesis Including Anosmia.

bioRxiv 2020 Aug 10. Epub 2020 Aug 10.

The ongoing COVID-19 pandemic is associated with substantial morbidity and mortality. While much has been learned in the first months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation and many patients with this finding show no or only minor respiratory signs. Studies in animals experimentally infected with SARS-CoV-2, the cause of COVID-19, provide opportunities to study aspects of the disease not easily investigated in human patients. COVID-19 severity ranges from asymptomatic to lethal. Most experimental infections provide insights into mild disease. Here, using K18-hACE2 mice that we originally developed for SARS studies, we show that infection with SARS-CoV-2 causes severe disease in the lung, and in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Further, we show that infusion of convalescent plasma (CP) from a recovered COVID-19 patient provided protection against lethal disease. Mice developed anosmia at early times after infection. Notably, while treatment with CP prevented significant clinical disease, it did not prevent anosmia. Thus K18-hACE2 mice provide a useful model for studying the pathological underpinnings of both mild and lethal COVID-19 and for assessing therapeutic interventions.
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http://dx.doi.org/10.1101/2020.08.07.242073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430569PMC
August 2020

3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV-infected mice.

Sci Transl Med 2020 08 3;12(557). Epub 2020 Aug 3.

Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.

Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.
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http://dx.doi.org/10.1126/scitranslmed.abc5332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574915PMC
August 2020

Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model.

Lab Invest 2020 11 27;100(11):1388-1399. Epub 2020 Jul 27.

Department of Internal Medicine and Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl or HCO was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl/HCO-free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.
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http://dx.doi.org/10.1038/s41374-020-0474-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578062PMC
November 2020

Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment.

Cell 2020 08 10;182(3):734-743.e5. Epub 2020 Jun 10.

State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510182, China; Institute of Infectious Disease, Guangzhou Eighth People's Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510060, China. Electronic address:

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.
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http://dx.doi.org/10.1016/j.cell.2020.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284240PMC
August 2020

Illuminating COVID-19 lung disease through autopsy studies.

EBioMedicine 2020 Jul 3;57:102865. Epub 2020 Jul 3.

Department of Pediatrics, Pappajohn Biomedical Institute, University of Iowa, Iowa City, IA 52240, United States. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2020.102865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332440PMC
July 2020

Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract.

bioRxiv 2020 Jun 1. Epub 2020 Jun 1.

Pathology, University of Iowa College of Medicine, University of Iowa, Iowa City, IA USA.

Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Despite numerous reports describing ACE2 mRNA abundance and tissue distribution, there remains a paucity of data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors. Here, we systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19. Our results reveal that ACE2 protein is highest within the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. However, ACE2 protein was increased in children, a demographic with a reduced incidence of severe COVID-19. These results offer new insights into ACE2 localization and function in susceptibility to COVID-19.
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http://dx.doi.org/10.1101/2020.04.22.056127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302220PMC
June 2020

Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection.

mBio 2020 04 7;11(2). Epub 2020 Apr 7.

Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans and remains endemic in the Middle East since first being identified in 2012. There are currently no approved vaccines or therapies available for MERS-CoV. In this study, we evaluated parainfluenza virus 5 (PIV5)-based vaccine expressing the MERS-CoV envelope spike protein (PIV5/MERS-S) in a human DPP4 knockin C57BL/6 congenic mouse model (hDPP4 KI). Following a single-dose intranasal immunization, PIV5-MERS-S induced neutralizing antibody and robust T cell responses in hDPP4 KI mice. A single intranasal administration of 10 PFU PIV5-MERS-S provided complete protection against a lethal challenge with mouse-adapted MERS-CoV (MERS6.1.2) and improved virus clearance in the lung. In comparison, single-dose intramuscular immunization with 10 PFU UV-inactivated MERS6.1.2 mixed with Imject alum provided protection to only 25% of immunized mice. Intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated MERS-CoV, suggestive of a hypersensitivity-type response. Overall, our study indicated that PIV5-MERS-S is a promising effective vaccine candidate against MERS-CoV infection. MERS-CoV causes lethal infection in humans, and there is no vaccine. Our work demonstrates that PIV5 is a promising vector for developing a MERS vaccine. Furthermore, success of PIV5-based MERS vaccine can be employed to develop a vaccine for emerging CoVs such as SARS-CoV-2, which causes COVID-19.
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http://dx.doi.org/10.1128/mBio.00554-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157776PMC
April 2020

Longitudinal phenotype development in a minipig model of neurofibromatosis type 1.

Sci Rep 2020 03 19;10(1):5046. Epub 2020 Mar 19.

Department of Radiology, University of Iowa, Iowa City, IA, USA.

Neurofibromatosis type 1 (NF1) is a rare, autosomal dominant disease with variable clinical presentations. Large animal models are useful to help dissect molecular mechanisms, determine relevant biomarkers, and develop effective therapeutics. Here, we studied a NF1 minipig model (NF1) for the first 12 months of life to evaluate phenotype development, track disease progression, and provide a comparison to human subjects. Through systematic evaluation, we have shown that compared to littermate controls, the NF1 model develops phenotypic characteristics of human NF1: [1] café-au-lait macules, [2] axillary/inguinal freckling, [3] shortened stature, [4] tibial bone curvature, and [5] neurofibroma. At 4 months, full body computed tomography imaging detected significantly smaller long bones in NF1 minipigs compared to controls, indicative of shorter stature. We found quantitative evidence of tibial bowing in a subpopulation of NF1 minipigs. By 8 months, an NF1 boar developed a large diffuse shoulder neurofibroma, visualized on magnetic resonance imaging, which subsequently grew in size and depth as the animal aged up to 20 months. The NF1 minipig model progressively demonstrates signature attributes that parallel clinical manifestations seen in humans and provides a viable tool for future translational NF1 research.
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http://dx.doi.org/10.1038/s41598-020-61251-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081358PMC
March 2020

Loss of iRhom2 accelerates fat gain and insulin resistance in diet-induced obesity despite reduced adipose tissue inflammation.

Metabolism 2020 05 2;106:154194. Epub 2020 Mar 2.

Inflammation Program, Roy J. and Lucille A. Carver College of Medicine University of Iowa, Iowa City, IA, USA; Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine University of Iowa, Iowa City, IA, USA; Immunology Graduate Program, Iowa City, IA, USA; Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, USA. Electronic address:

Background: Low-grade inflammation and metabolic dysregulation are common comorbidities of obesity, both of which are associated with alterations in iRhom2-regulated pro-inflammatory cytokine and epidermal growth factor receptor (EGFR) ligand signaling.

Objective: Our objective was to determine the role of iRhom2 in the regulation of low-grade inflammation and metabolic dysregulation in a murine model of diet-induced obesity.

Methods: Wild type (WT) and iRhom2-deficient mice were fed normal chow (NC) or a high-fat diet (HFD) starting at 5 weeks of age for up to 33 weeks. Body composition, glucose and insulin tolerance, feeding behavior, and indirect calorimetry were measured at defined time points. Adipose tissue cytokine expression and inflammatory lesions known as crown-like structures (CLS) were analyzed at the end-point of the study.

Results: iRhom2-deficient mice show accelerated fat gain on a HFD, accompanied by insulin resistance. Indirect calorimetry did not demonstrate changes in energy expenditure or food intake, but locomotor activity was significantly reduced in HFD iRhom2-deficient mice. Interestingly, CLS, macrophage infiltration, and tumor necrosis factor (TNF) production were decreased in adipose tissue from HFD iRhom2-deficient mice, but circulating cytokines were unchanged. In inguinal and perigonadal fat, the EGFR ligand amphiregulin was markedly induced in HFD controls but completely prevented in iRhom2-deficient mice, suggesting a potentially dominant role of EGFR-dependent mechanisms over TNF in the modulation of insulin sensitivity.

Conclusions: This study elucidates a novel role for iRhom2 as an immuno-metabolic regulator that affects adipose tissue inflammation independent of insulin resistance.
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http://dx.doi.org/10.1016/j.metabol.2020.154194DOI Listing
May 2020

Evolving challenges to model human diseases for translational research.

Cell Tissue Res 2020 May 4;380(2):305-311. Epub 2020 Mar 4.

Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA, USA.

Animal models are a significant component of biomedical research and play an important role in translational studies. Traditionally, rodent models have been the mainstay and principal choice of researchers but in recent years, there have been significant changes in the landscape of animal modeling. For example, newer techniques have greatly expanded the use and successful application of large animal models such as pigs for translational studies. The evolving types and species of animal models can influence the research landscape in terms of facilities, expertise, reproducibility and funding streams, which creates new challenges for research studies. It is also important that investigators are prepared to address the necessity of their animal model research and capable to educate the public regarding its value.
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http://dx.doi.org/10.1007/s00441-019-03134-3DOI Listing
May 2020

RABL6A Is an Essential Driver of MPNSTs that Negatively Regulates the RB1 Pathway and Sensitizes Tumor Cells to CDK4/6 Inhibitors.

Clin Cancer Res 2020 06 21;26(12):2997-3011. Epub 2020 Feb 21.

Molecular Medicine Graduate Program, University of Iowa, Iowa City, Iowa.

Purpose: Malignant peripheral nerve sheath tumors (MPNST) are deadly sarcomas that lack effective therapies. In most MPNSTs, the retinoblastoma (RB1) tumor suppressor is disabled by hyperactivation of cyclin-dependent kinases (CDK), commonly through loss of CDK-inhibitory proteins such as p27(Kip1). RABL6A is an inhibitor of RB1 whose role in MPNSTs is unknown. To gain insight into MPNST development and establish new treatment options, we investigated RABL6A-RB1 signaling and CDK inhibitor-based therapy in MPNSTs.

Experimental Design: We examined patient-matched MPNSTs and precursor lesions by RNA sequencing (RNA-Seq) and IHC. Molecular and biological effects of silencing RABL6A and/or p27 in MPNST lines and normal human Schwann cells were determined. Tumor-suppressive effects of CDK inhibitors were measured in MPNST cells and orthotopic tumors.

Results: RABL6A was dramatically upregulated in human MPNSTs compared with precursor lesions, which correlated inversely with p27 levels. Silencing RABL6A caused MPNST cell death and G arrest that coincided with p27 upregulation, CDK downregulation, and RB1 activation. The growth-suppressive effects of RABL6A loss, and its regulation of RB1, were largely rescued by p27 depletion. Importantly, reactivation of RB1 using a CDK4/6 inhibitor (palbociclib) killed MPNST cells in an RABL6A-dependent manner and suppressed MPNST growth . Low-dose combination of drugs targeting multiple RB1 kinases (CDK4/6, CDK2) had enhanced antitumorigenic activity associated with potential MPNST cell redifferentiation.

Conclusions: RABL6A is a new driver of MPNST pathogenesis that acts in part through p27-RB1 inactivation. Our results suggest RB1 targeted therapy with multiple pathway drugs may effectively treat MPNSTs.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299809PMC
June 2020

Validating indicators of CNS disorders in a swine model of neurological disease.

PLoS One 2020 19;15(2):e0228222. Epub 2020 Feb 19.

Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, South Dakota, United States of America.

Genetically modified swine disease models are becoming increasingly important for studying molecular, physiological and pathological characteristics of human disorders. Given the limited history of these model systems, there remains a great need for proven molecular reagents in swine tissue. Here, to provide a resource for neurological models of disease, we validated antibodies by immunohistochemistry for use in examining central nervous system (CNS) markers in a recently developed miniswine model of neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant tumor predisposition disorder stemming from mutations in NF1, a gene that encodes the Ras-GTPase activating protein neurofibromin. Patients classically present with benign neurofibromas throughout their bodies and can also present with neurological associated symptoms such as chronic pain, cognitive impairment, and behavioral abnormalities. As validated antibodies for immunohistochemistry applications are particularly difficult to find for swine models of neurological disease, we present immunostaining validation of antibodies implicated in glial inflammation (CD68), oligodendrocyte development (NG2, O4 and Olig2), and neuron differentiation and neurotransmission (doublecortin, GAD67, and tyrosine hydroxylase) by examining cellular localization and brain region specificity. Additionally, we confirm the utility of anti-GFAP, anti-Iba1, and anti-MBP antibodies, previously validated in swine, by testing their immunoreactivity across multiple brain regions in mutant NF1 samples. These immunostaining protocols for CNS markers provide a useful resource to the scientific community, furthering the utility of genetically modified miniswine for translational and clinical applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228222PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029865PMC
April 2020

Histopathologic Evaluation and Scoring of Viral Lung Infection.

Methods Mol Biol 2020 ;2099:205-220

Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA.

Emergent coronaviruses such as MERS-CoV and SARS-CoV can cause significant morbidity and mortality in infected individuals. Lung infection is a common clinical feature and contributes to disease severity as well as viral transmission. Animal models are often required to study viral infections and therapies, especially during an initial outbreak. Histopathology studies allow for identification of lesions and affected cell types to better understand viral pathogenesis and clarify effective therapies. Use of immunostaining allows detection of presumed viral receptors and viral tropism for cells can be evaluated to correlate with lesions. In the lung, lesions and immunostaining can be qualitatively described to define the cell types, microanatomic location, and type of changes seen. These features are important and necessary, but this approach can have limitations when comparing treatment groups. Semiquantitative and quantitative tissue scores are more rigorous as these provide the ability to statistically compare groups and increase the reproducibility and rigor of the study. This review describes principles, approaches, and resources that can be useful to evaluate coronavirus lung infection, focusing on MER-CoV infection as the principal example.
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http://dx.doi.org/10.1007/978-1-0716-0211-9_16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7123785PMC
September 2020

Pre-existing neutralizing antibodies prevent CD8 T cell-mediated immunopathology following respiratory syncytial virus infection.

Mucosal Immunol 2020 05 16;13(3):507-517. Epub 2019 Dec 16.

Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA, 52242, USA.

Despite being a leading cause of severe respiratory disease, there remains no licensed respiratory syncytial virus (RSV) vaccine. Neutralizing antibodies reduce the severity of RSV-associated disease, but are not sufficient for preventing reinfection. In contrast, the role of memory CD8 T cells in protecting against a secondary RSV infection is less established. We recently demonstrated that high-magnitude memory CD8 T cells efficiently reduced lung viral titers following RSV infection, but induced fatal immunopathology that was mediated by IFN-γ. To evaluate the ability of RSV-specific neutralizing antibodies to prevent memory CD8 T cell-mediated immunopathology, mice with high-magnitude memory CD8 T cell responses were treated with neutralizing antibodies prior to RSV challenge. Neutralizing antibody treatment significantly reduced morbidity and prevented mortality following RSV challenge compared with IgG-treated controls. Neutralizing antibody treatment restricted early virus replication, which caused a substantial reduction in memory CD8 T cell activation and IFN-γ production, directly resulting in survival. In contrast, therapeutic neutralizing antibody administration did not impact morbidity, mortality, or IFN-γ levels, despite significantly reducing lung viral titers. Therefore, only pre-existing neutralizing antibodies prevent memory CD8 T cell-mediated immunopathology following RSV infection. Overall, our results have important implications for the development of future RSV vaccines.
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http://dx.doi.org/10.1038/s41385-019-0243-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181396PMC
May 2020

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia.

Nat Commun 2019 10 28;10(1):4906. Epub 2019 Oct 28.

Department of Pediatrics, Pappajohn Biomedical Institute, University of Iowa, Iowa City, IA, 52242, USA.

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSA mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.
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http://dx.doi.org/10.1038/s41467-019-12922-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817825PMC
October 2019

An investigation of p53 in skeletal muscle aging.

J Appl Physiol (1985) 2019 10 29;127(4):1075-1084. Epub 2019 Aug 29.

Departments of Internal Medicine and Molecular Physiology and Biophysics and Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, Iowa.

Age-related skeletal muscle atrophy is a very common and serious condition that remains poorly understood at the molecular level. Several lines of evidence have suggested that the tumor suppressor p53 may play a central, causative role in skeletal muscle aging, whereas other, apparently contradictory lines of evidence have suggested that p53 may be critical for normal skeletal muscle function. To help address these issues, we performed an aging study in male muscle-specific p53-knockout mice (p53 mKO mice), which have a lifelong absence of p53 expression in skeletal muscle fibers. We found that the absence of p53 expression in skeletal muscle fibers had no apparent deleterious or beneficial effects on skeletal muscle mass or function under basal conditions up to 6 mo of age, when mice are fully grown and exhibit peak muscle mass and function. Furthermore, at 22 and 25 mo of age, when age-related muscle weakness and atrophy are clearly evident in mice, p53 mKO mice demonstrated no improvement or worsening of skeletal muscle mass or function relative to littermate control mice. At advanced ages, p53 mKO mice began to die prematurely and had an increased incidence of osteosarcoma, precluding analyses of muscle mass and function in very old p53 mKO mice. In light of these results, we conclude that p53 expression in skeletal muscle fibers has minimal if any direct, cell autonomous effect on basal or age-related changes in skeletal muscle mass and function up to at least 22 mo of age. Previous studies implicated the transcriptional regulator p53 as a potential mediator of age-related skeletal muscle weakness and atrophy. We tested this hypothesis by investigating the effect of aging in muscle-specific p53-knockout mice. Our results strongly suggest that p53 activity within skeletal muscle fibers is not required for age-related skeletal muscle atrophy or weakness.
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http://dx.doi.org/10.1152/japplphysiol.00363.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850986PMC
October 2019

IFN-I response timing relative to virus replication determines MERS coronavirus infection outcomes.

J Clin Invest 2019 07 29;129(9):3625-3639. Epub 2019 Jul 29.

Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.

Type 1 IFNs (IFN-I) generally protect mammalian hosts from virus infections, but in some cases, IFN-I is pathogenic. Because IFN-I is protective, it is commonly used to treat virus infections for which no specific approved drug or vaccine is available. The Middle East respiratory syndrome-coronavirus (MERS-CoV) is such an infection, yet little is known about the role of IFN-I in this setting. Here, we show that IFN-I signaling is protective during MERS-CoV infection. Blocking IFN-I signaling resulted in delayed virus clearance, enhanced neutrophil infiltration, and impaired MERS-CoV-specific T cell responses. Notably, IFN-I administration within 1 day after infection (before virus titers peak) protected mice from lethal infection, despite a decrease in IFN-stimulated gene (ISG) and inflammatory cytokine gene expression. In contrast, delayed IFN-β treatment failed to effectively inhibit virus replication, increased infiltration and activation of monocytes, macrophages, and neutrophils in the lungs, and enhanced proinflammatory cytokine expression, resulting in fatal pneumonia in an otherwise sublethal infection. Together, these results suggest that the relative timing of the IFN-I response and maximal virus replication is key in determining outcomes, at least in infected mice. By extension, IFN-αβ or combination therapy may need to be used cautiously to treat viral infections in clinical settings.
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http://dx.doi.org/10.1172/JCI126363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715373PMC
July 2019

An Introduction to Pathology in Biomedical Research: A Mission-Critical Specialty for Reproducibility and Rigor in Translational Research.

ILAR J 2018 12;59(1):1-3

GEMpath Inc. Longmont, Colorado.

This issue of ILAR Journal focusses on pathology and pathologists in biomedical research, more specifically in preclinical translational research involving (nonhuman) animals, emphasizing academic settings. Considerations in study design and planning to maximize benefit from pathologists and pathology resources are reviewed. Adjunctive technologies including molecular techniques, digital pathology, and imaging are highlighted. Additional considerations regarding safety and regulatory concerns, and veterinary clinical trials are reviewed as well. Pathology has been fundamental to understanding clinical disease, remains fundamental to diagnosing disease, and is required in drug and device development. Broader integration of pathology expertise and well-designed pathology investigations have much to offer research rigor and reproducibility, and successful translation from biomedical research.
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http://dx.doi.org/10.1093/ilar/ilz008DOI Listing
December 2018

Assessment of nociception and related quality-of-life measures in a porcine model of neurofibromatosis type 1.

Pain 2019 11;160(11):2473-2486

Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD, United States.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder resulting from germline mutations in the NF1 gene, which encodes neurofibromin. Patients experience a variety of symptoms, but pain in the context of NF1 remains largely underrecognized. Here, we characterize nociceptive signaling and pain behaviors in a miniswine harboring a disruptive NF1 mutation (exon 42 deletion). We present the first characterization of pain-related behaviors in a pig model of NF1, identifying unchanged agitation scores, lower tactile thresholds (allodynia), and decreased response latencies to thermal laser stimulation (hyperalgesia) in NF1 (females only) pigs. Male NF1 pigs with tumors showed reduced sleep quality and increased resting, 2 health-related quality-of-life symptoms found to be comorbid in people with NF1 pain. We explore these phenotypes in relationship to suppression of the increased activity of the N-type voltage-gated calcium (CaV2.2) channel by pharmacological antagonism of phosphorylation of a regulatory protein-the collapsin response mediator protein 2 (CRMP2), a known interactor of neurofibromin, and by targeting the interface between the α subunit of CaV2.2 and the accessory β-subunits with small molecules. Our data support the use of NF1 pigs as a large animal model for studying NF1-associated pain and for understanding the pathophysiology of NF1. Our findings demonstrate the translational potential of 2 small molecules in reversing ion channel remodeling seen in NF1. Interfering with CaV2.2, a clinically validated target for pain management, might also be a promising therapeutic strategy for NF1-related pain management.
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http://dx.doi.org/10.1097/j.pain.0000000000001648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800626PMC
November 2019