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The Golden Syrian hamster is a well-characterized rodent model for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2)-associated pneumonia. We sought to characterize the pulmonary disease course during SARS-CoV-2 infection (strain USA-WA1/2020) in the hamster model using micro-computed tomography (micro-CT) and compare radiologic observations with histopathologic findings. We observed a range of radiologic abnormalities, including ground glass opacities (GGOs), consolidations, air bronchograms, and pneumomediastinum. The appearance, distribution, and progression of these abnormalities in hamsters were similar to those observed in the lungs of coronavirus disease 2019 (COVID-19) patients by clinical CT and chest X-rays, and correlated with clinical signs and weight loss during the course of disease. Histopathological analysis of infected hamsters revealed lung pathology characteristic of COVID-19 pneumonia, and we observed a strong association between CT and histopathologic scorings. We also analyzed accumulation of air in the thoracic cavity by both manual and automated threshold-based segmentation and found that automated analysis significantly decreases the time needed for data analysis. Data presented here demonstrate that micro-CT imaging can be a major tool in preclinical investigative studies using animal models by providing early and detailed assessment of disease severity and outcomes., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: All authors are current or former employees of Pfizer Inc., and may, therefore, be shareholders.

Several chemicals and pharmaceuticals increase the incidence of hemangiosarcomas (HSAs) in mice, but the relevance to humans is uncertain. Recently, canine HSAs were identified as a powerful tool for investigating the pathogenesis of human HSAs. To characterize the cellular phenotype of canine HSAs, we evaluated immunoreactivity and/or messenger RNA (mRNA) expression of markers for hematopoietic stem cells (HSCs), endothelial cells (ECs), a tumor suppressor protein, and a myeloid marker in canine HSAs. Neoplastic canine cells expressed EC markers and a myeloid marker, but expressed HSC markers less consistently. The canine tumor expression results were then compared to previously published immunoreactivity results for these markers in human and mouse HSAs. There are 2 noteworthy differences across species: (1) most human HSAs had HSC marker expression, indicating that they were comprised of tumor cells that were less differentiated than those in canine and mouse tumors; and (2) human and canine HSAs expressed a late-stage EC maturation marker, whereas mouse HSAs were negative, suggesting that human and canine tumors may retain greater differentiation potential than mouse tumors. These results indicate that HSA development is variable across species and that caution is necessary when discussing translation of carcinogenic risk from animal models to humans.

Myeloperoxidase (MPO) is a highly abundant protein within the neutrophil that is associated with lipoprotein oxidation, and increased plasma MPO levels are correlated with poor prognosis after myocardial infarct. Thus, MPO inhibitors have been developed for the treatment of heart failure and acute coronary syndrome in humans. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide PF-06282999 is a recently described selective small molecule mechanism-based inactivator of MPO. Here, utilizing PF-06282999, we investigated the role of MPO to regulate atherosclerotic lesion formation and composition in the Ldlr-/- mouse model of atherosclerosis. Though MPO inhibition did not affect lesion area in Ldlr-/- mice fed a Western diet, reduced necrotic core area was observed in aortic root sections after MPO inhibitor treatment. MPO inhibition did not alter macrophage content in and leukocyte homing to atherosclerotic plaques. To assess non-invasive monitoring of plaque inflammation, [18F]-Fluoro-deoxy-glucose (FDG) was administered to Ldlr-/- mice with established atherosclerosis that had been treated with clinically relevant doses of PF-06282999, and reduced FDG signal was observed in animals treated with a dose of PF-06282999 that corresponded with reduced necrotic core area. These data suggest that MPO inhibition does not alter atherosclerotic plaque area or leukocyte homing, but rather alters the inflammatory tone of atherosclerotic lesions; thus, MPO inhibition could have utility to promote atherosclerotic lesion stabilization and prevent atherosclerotic plaque rupture., Competing Interests: The authors are employees of Pfizer, Inc. and own Pfizer stock. The studies were funded by Pfizer, Inc. The compound described in this manuscript is patented under US patent 8,835,449. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Pharmaceuticals and chemicals produce hemangiosarcomas (HS) in mice, often by nongenotoxic, proliferative mechanisms. A mode-of-action (MOA) for hemangiosarcoma was proposed based on information presented at an international workshop (Cohen et al., Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18.). Five key elements of the MOA were articulated and included hypoxia, macrophage activation, increased angiogenic growth factors, dysregulated angiogenesis/erythropoiesis, and endothial cell proliferation. The goal of the current study was to add to the weight-of-evidence for the proposed MOA by assessing these key elements with 3 different compounds of varying potency for HS induction: fenretinide (high), troglitazone (intermediate), and elmiron (low). Multiple endpoints, including hypoxia (hyproxyprobe, transcriptomics), endothelial cell (EC) proliferation, and clinical and anatomic pathology, were assessed after 2, 4, and 13-weeks of treatment in B6C3F1 mice. All 3 compounds demonstrated strong evidence for dysregulated erythropoiesis (decrease in RBC and a failure to increase reticulocytes) and macrophage activation (4- to 11-fold increases); this pattern of hematological changes in mice might serve as an early biomarker to evaluate EC proliferation in suspected target organs for potential HS formation. Fenretinide demonstrated all 5 key elements, while troglitazone demonstrated 4 and elmiron demonstrated 3. Transcriptomics provided support for the 5 elements of the MOA, but was not any more sensitive than hypoxyprobe immunohistochemistry for detecting hypoxia. The overall transcriptional evidence for the key elements of the proposed MOA was also consistent with the potency of HS induction. These data, coupled with the previous work with 2-butoxyethanol and pregablin, increase the weight-of-evidence for the proposed MOA for HS formation., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

A number of chemical compounds have been shown to induce liver tumors in mice but not in other species. While several mechanisms for this species-specific tumorigenicity have been proposed, no definitive mechanism has been established. We examined the effects of the nongenotoxic rodent hepatic carcinogen, WY-14,643, in male mice from a high liver tumor susceptible strain (C3H/HeJ), and from a low tumor susceptible strain (C57BL/6). WY-14,643, a PPARα activator induced widespread increases in the expression of some endogenous retroelements, namely members of LTR and LINE elements in both strains. The expression of a number of known retroviral defense genes was also elevated. We also demonstrated that basal immune-mediated viral defense was elevated in C57BL/6 mice (the resistant strain) and that WY-14,643 further activated those immuno-defense processes. We propose that the previously reported >100X activity of retroelements in mice drives mouse-specific tumorigenicity. We also propose that C57BL/6's competent immune to retroviral activation allows it to remove cells before the activation of these elements can result in significant chromosomal insertions and mutation. Finally, we showed that WY-14,643 treatment induced gene signatures of DNA recombination in the sensitive C3H/HeJ strain.

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK β1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the β 1 subunit. After confirming that human and rodent kidney predominately express AMPK β 1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles(®)). Fluorescence of the Bioparticles(®) is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles(®) phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS(®) Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles(®). The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles(®). In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development.

Treatment-induced epididymal inflammation and granuloma formation is only an occasional problem in preclinical drug development, but it can effectively terminate the development of that candidate molecule. Screening for backup molecules without that toxicity must be performed in animals (generally rats) that requires at least 2 to 3 weeks of in vivo exposure, a great deal of specially synthesized candidate compound, and histologic examination of the target tissues. We instead hypothesized that these treatments induced proinflammatory gene expression, and so used mixed-cell cultures from the rat epididymal tubule to monitor the induction of proinflammatory cytokines. Cells were exposed for 24 hr and then cytotoxicity was evaluated with the MTS assay and mRNA levels of Interleukin-6 (IL-6) and growth-related oncogene (GRO) were measured. We found that compounds that were more toxic in vivo stimulated a greater induction of IL-6 and GRO mRNA levels in vitro. By relating effective concentrations in vitro with the predicted C(eff), we could rank compounds by their propensity to induce inflammation in rats in vivo. This method allowed the identification of several compounds with very low inflammatory induction in vitro. When tested in rats, the compounds produced small degrees of inflammation at an acceptable margin (approximately 20×), and have progressed into further development., (© 2014 Wiley Periodicals, Inc.)

A number of immunomodulatory therapeutics increase the risk of disease associated with latent herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), a member of the lymphocryptovirus (LCV) family that infects humans. The diseases associated with loss of immunity to these viruses can have major impacts on patients as well as on the commercial viability of the immunomodulatory therapeutics. In an effort to develop non-clinical methods for measuring effects on anti-viral immunity, we have developed an interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) assay to quantify the number of CMV or LCV-reactive T-cells in peripheral blood of cynomolgus macaques. After optimization of various parameters, the IFN-γ ELISPOT assay was characterized for specificity, intra-assay, monkey-to-monkey, and longitudinal variability and sensitivity to immunosuppression. The results show that nearly all animals have detectable responses against both CMV and LCV and responses were derived from T-cells specific to the virus of interest. Analyses of variability show assay reproducibility (≤23% CV), and that variability over time in anti-viral responses in individual animals (larger for LCV than for CMV) was ∼2-fold in most animals over a 3-month time period, which is predicted to allow for detection of drug-induced changes when using group sizes typical of non-clinical studies. In addition, the IFN-γ ELISPOT assay was capable of detecting decreases in the numbers of CMV and LCV reactive T-cells induced by immunosuppressive drugs in vitro. This assay may allow for non-clinical assessment of the effects of immunomodulatory therapeutics on anti-viral T-cell immunity in monkeys, and may help determine if therapeutics increase the risk of reactivating latent viral infections.

The phosphodiesterases (PDEs) are a superfamily of enzymes that regulate spatio-temporal signaling by the intracellular second messengers cAMP and cGMP. PDE2A is expressed at high levels in the mammalian brain. To advance our understanding of the role of this enzyme in regulation of neuronal signaling, we here describe the distribution of PDE2A in the rat brain. PDE2A mRNA was prominently expressed in glutamatergic pyramidal cells in cortex, and in pyramidal and dentate granule cells in the hippocampus. Protein concentrated in the axons and nerve terminals of these neurons; staining was markedly weaker in the cell bodies and proximal dendrites. In addition, in both hippocampus and cortex, small populations of non-pyramidal cells, presumed to be interneurons, were strongly immunoreactive. PDE2A mRNA was expressed in medium spiny neurons in neostriatum. Little immunoreactivity was observed in cell bodies, whereas dense immunoreactivity was found in the axon tracts of these neurons and their terminal regions in globus pallidus and substantia nigra pars reticulata. Immunostaining was dense in the medial habenula, but weak in other diencephalic regions. In midbrain and hindbrain, immunostaining was restricted to discrete regions of the neuropil or clusters of cell bodies. These results suggest that PDE2A may modulate cortical, hippocampal and striatal networks at several levels. Preferential distribution of PDE2A into axons and terminals of the principal neurons suggests roles in regulation of axonal excitability or transmitter release. The enzyme is also in forebrain interneurons, and in mid- and hindbrain neurons that may modulate forebrain networks and circuits., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72-96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.

Phosphodiesterase 5A (PDE5A) inhibitors improve functional recovery after middle cerebral artery occlusion (MCA-o) in rats. We used the PDE5A inhibitor 3-(4-(2-hydroxyethyl)piperazin-1-yl)-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one hydrochloride (PF-5) to determine the timing, duration, and degree of inhibition that yields maximum efficacy. We also investigated the localization of PDE5A to determine the tissues and cells that would be targets for PDE5 inhibition and that may mediate efficacy. Nearly complete inhibition of PDE5A, starting 24 h after MCA-o and continued for 7 days, resulted in nearly complete recovery of sensorimotor function that was sustained for 3 months. Delaying administration until 72 h after MCA-o resulted in equivalent efficacy, whereas delaying treatment for 14 days was ineffective. Treatment for 7 days was equivalently efficacious to 28 or 84 days of treatment, whereas treatment for 1 day was less effective. In the normal forebrain, PDE5A immunoreactivity was prominent in smooth muscle of meningeal arteries and a few smaller blood vessels, with weak staining in a few widely scattered cortical neurons and glia. At 24 and 48 h after MCA-o, the number and intensity of blood vessel staining increased in the infarcted cortex and striatum. PDE5A immunoreactivity also was increased at 48 h in putative microglia in penumbra, whereas there was no change in staining of the scattered cortical neurons. Given the window for efficacy and the PDE5A distribution, we hypothesize that efficacy results from an effect on vasculature, and perhaps modulation of microglial function, both of which may facilitate recovery of neuronal function.

Phosphodiesterases (PDEs) comprise a family of enzymes that regulate the levels of cyclic nucleotides, key second messengers that mediate a diverse array of functions. PDE2A is an evolutionarily conserved cGMP-stimulated cAMP and cGMP PDE. In the present study, the regional and cellular distribution of PDE2A in tissues of rats, mice, cynomolgus monkeys, dogs, and humans was evaluated by immunohistochemistry. A polyclonal antibody directed to the C-terminal portion of PDE2A specifically detected PDE2A by Western blotting and by immunohistochemistry. The pattern of PDE2A immunoreactivity (ir) was consistent across all species. Western blot analysis demonstrated that PDE2A was most abundant in the brain relative to peripheral tissues. PDE2A ir was heterogeneously distributed within brain and was selectively expressed in particular peripheral tissues. In the brain, prominent immunoreactivity was apparent in components of the limbic system, including the isocortex, hippocampus, amygdala, habenula, basal ganglia, and interpeduncular nucleus. Cytoplasmic PDE2A staining was prominent in several peripheral tissues, including the adrenal zona glomerulosa, neurons of enteric ganglia, endothelial cells in all organs, lymphocytes of spleen and lymph nodes, and pituitary. These studies suggest that PDE2A is evolutionarily conserved across mammalian species and support the hypothesis that the enzyme plays a fundamental role in signal transduction.

A monoclonal antibody directed against the amino terminal of rat phosphodiesterase 10A (PDE10A) was used to localize PDE10A in multiple central nervous system (CNS) and peripheral tissues from mouse, rat, dog, cynomolgus macaque, and human. PDE10A immunoreactivity is strongly expressed in the CNS of these species with limited expression in peripheral tissues. Within the brain, strong immunoreactivity is present in both neuronal cell bodies and neuropil of the striatum, in striatonigral and striatopallidal white matter tracks, and in the substantia nigra and globus pallidus. Outside the brain, PDE10A immunoreactivity is less intense, and distribution is limited to few tissues such as the testis, epididymal sperm, and enteric ganglia. These data demonstrate that PDE10A is an evolutionarily conserved phosphodiesterase highly expressed in the brain but with restricted distribution in the periphery in multiple mammalian species.

PDE10A is a newly identified cAMP/cGMP phosphodiesterase for which mRNA is highly expressed in the mammalian striatum. In the present study, PDE10A protein and mRNA expression throughout the rat brain were determined, using a monoclonal antibody (24F3.F11) for Western blot and immunohistochemical analyses and an antisense riboprobe for in situ hybridization. High levels of mRNA are observed in most of the neuronal cell bodies of striatal complex (caudate n, n. accumbens and olfactory tubercle), indicating that PDE10A is expressed by the striatal medium spiny neurons. PDE10A-like immunoreactivity is dense throughout the striatal neuropil, as well as in the internal capsule, globus pallidus, and substantia nigra. These latter regions lack significant expression of PDE10A mRNA. Thus, PDE10A is transported throughout the dendritic tree and down the axons to the terminals of the medium spiny neurons. These data suggest a role for PDE10A in regulating activity within both the striatonigral and striatopallidal pathways. In addition, PDE10A immunoreactivity and mRNA are found at lower levels in the hippocampal pyramidal cell layer, dentate granule cell layer and throughout the cortex and cerebellar granule cell layer. Immunoreactivity is detected only in cell bodies in these latter regions. This more restricted subcellular localization of PDE10A outside the striatum suggests a second, distinct function for the enzyme in these regions.