Investigation of Various Intramuscular Volumes Delivered to the Semimembranosus Muscle of .

The goal of this study is to provide quantitative data on the ideal volume for intramuscular (IM) injections into the semimembranosus muscle of guinea pigs weighing between 320 to 410 grams. This evaluation comprised 2 experiments. The first was to assess dispersion leakage of intramuscularly injected iohexol, a radiocontrast agent commonly used in Computed Tomography (CT), based on analysis of in vivo imaging.

Characterization of Brain Inflammation, Apoptosis, Hypoxia, Blood-Brain Barrier Integrity and Metabolism in Venezuelan Equine Encephalitis Virus (VEEV TC-83) Exposed Mice by In Vivo Positron Emission Tomography Imaging.

Traditional pathogenesis studies of alphaviruses involves monitoring survival, viremia, and pathogen dissemination via serial necropsies; however, molecular imaging shifts this paradigm and provides a dynamic assessment of pathogen infection. Positron emission tomography (PET) with PET tracers targeted to study neuroinflammation (,-diethyl-2-[4-phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide, [F]DPA-714), apoptosis (caspase-3 substrate, [F]CP-18), hypoxia (fluormisonidazole, [F]FMISO), blood-brain barrier (BBB) integrity ([F]albumin), and metabolism (fluorodeoxyglucose, [F]FDG) was performed on C3H/HeN mice infected intranasally with 7000 plaque-forming units (PFU) of Venezuelan equine encephalitis virus (VEEV) TC-83. The main findings are as follows: (1) whole-brain [F]DPA-714 and [F]CP-18 uptake increased three-fold demonstrating, neuroinflammation and apoptosis, respectively; (2) [F]albumin uptake increased by 25% across the brain demonstrating an altered BBB; (3) [F]FMISO uptake increased by 50% across the whole brain indicating hypoxic regions; (4) whole-brain [F]FDG uptake was unaffected; (5) [F]DPA-714 uptake in (a) cortex, thalamus, striatum, hypothalamus, and hippocampus increased through day seven and decreased by day 10 post exposure, (b) olfactory bulb increased at day three, peaked day seven, and decreased day 10, and (c) brain stem and cerebellum increased through day 10.

Synthesis of [F]Favipiravir and Biodistribution in C3H/HeN Mice as Assessed by Positron Emission Tomography.

Favipiravir (T705; 6-fluoro-3-hydroxypyrazine-2-carboxamide) is a pyrazine analog that has demonstrated potent antiviral activity against a broad spectrum of viruses in multiple in vivo disease models. To better understand the compounds anti-viral activity, assessment of the drug's biodistribution and kinetics in vivo may lend insight into how best to evaluate the compound efficacy preclinically and to contribute to the design of clinical studies to take into account the compound's pharmacokinetic distribution and kinetics. In the current study, a method for synthesis of [F]favipiravir was developed and the biodistribution in mice naïve to and pre-dosed with favipiravir was assessed by PET and gamma counting of tissue samples.

Bioengineering of bacterial pathogens for noninvasive imaging and in vivo evaluation of therapeutics.

Critical bacterial pathogens of public health and biodefense concerns were engineered to constitutively express Escherichia coli enzyme thymidine kinase (TK) that allows for noninvasive nuclear imaging via phosphorylation and entrapment of radiolabeled nucleoside analog 1-(2'deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-iodouracil (FIAU). Expression of functional TK was established using a nucleoside analog Zidovudine that impeded the growth of tk-engineered bacteria. Significantly, no observable growth differences were detected for FIAU.

Countering Zika Virus: The USAMRIID Response.

The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) possesses an array of expertise in diverse capabilities for the characterization of emerging infectious diseases from the pathogen itself to human or animal infection models. The recent Zika virus (ZIKV) outbreak was a challenge and an opportunity to put these capabilities to work as a cohesive unit to quickly respond to a rapidly developing threat. Next-generation sequencing was used to characterize virus stocks and to understand the introduction and spread of ZIKV in the United States.

Evaluation of Volume of Intramuscular Injection into the Caudal Thigh Muscles of Female and Male BALB/c Mice ().

This study presents recommendations for intramuscular injection into the caudal thigh muscle of mice according to analysis of in vivo imaging of intramuscularly injected iohexol, a radiocontrast agent commonly used in CT imaging. An experienced laboratory animal technician using a Hamilton syringe intramuscularly injected iohexol into isoflurane-anesthetized female and male BALB/c mice. Injected volumes (25, 50, 100, and 200 μL) underwent CT scanning at 9 time points over a 3-h period.

Efficacy of favipiravir (T-705) in nonhuman primates infected with Ebola virus or Marburg virus.

Favipiravir is a broad-spectrum antiviral agent that has demonstrated efficacy against Ebola virus (EBOV) in rodents. However, there are no published reports of favipiravir efficacy for filovirus infection of nonhuman primates (NHPs). Here we evaluated the pharmacokinetic profile of favipiravir in NHPs, as well as in vivo efficacy against two filoviruses, EBOV and Marburg virus (MARV).

Intracellular conversion and in vivo dose response of favipiravir (T-705) in rodents infected with Ebola virus.

During the 2013-2016 Ebola virus (EBOV) outbreak in West Africa, our team at USAMRIID evaluated the antiviral activity of a number of compounds, including favipiravir (T-705), in vitro and in mouse and nonhuman primate (NHP) models of Ebola virus disease. In this short communication, we present our findings for favipiravir in cell culture and in mice, while an accompanying paper presents the results of NHP studies. We confirmed previous reports that favipiravir has anti-EBOV activity in mice.

[F]DPA-714 PET Imaging Reveals Global Neuroinflammation in Zika Virus-Infected Mice.

Purpose: The association of Zika virus (ZIKV) infection and development of neurological sequelae require a better understanding of the pathogenic mechanisms causing severe disease. The purpose of this study was to evaluate the ability and sensitivity of positron emission tomography (PET) imaging using [F]DPA-714, a translocator protein (TSPO) 18 kDa radioligand, to detect and quantify neuroinflammation in ZIKV-infected mice.

Procedures: We assessed ZIKV-induced pathogenesis in wild-type C57BL/6 mice administered an antibody to inhibit type I interferon (IFN) signaling.

Neuropathogenesis of Zika Virus in a Highly Susceptible Immunocompetent Mouse Model after Antibody Blockade of Type I Interferon.

Animal models are needed to better understand the pathogenic mechanisms of Zika virus (ZIKV) and to evaluate candidate medical countermeasures. Adult mice infected with ZIKV develop a transient viremia, but do not demonstrate signs of morbidity or mortality. Mice deficient in type I or a combination of type I and type II interferon (IFN) responses are highly susceptible to ZIKV infection; however, the absence of a competent immune system limits their usefulness for studying medical countermeasures.

Applications of in vivo imaging in the evaluation of the pathophysiology of viral and bacterial infections and in development of countermeasures to BSL3/4 pathogens.

While preclinical and clinical imaging have been applied to drug discovery/development and characterization of disease pathology, few examples exist where imaging has been used to evaluate infectious agents or countermeasures to biosafety level (BSL)3/4 threat agents. Viruses engineered with reporter constructs, i.e.

Design and selection parameters to accelerate the discovery of novel central nervous system positron emission tomography (PET) ligands and their application in the development of a novel phosphodiesterase 2A PET ligand.

To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB).

The synthesis and in vivo evaluation of [18F]PF-9811: a novel PET ligand for imaging brain fatty acid amide hydrolase (FAAH).

Introduction: Fatty acid amide hydrolase (FAAH) is responsible for the enzymatic degradation of the fatty acid amide family of signaling lipids, including the endogenous cannabinoid (endocannabinoid) anandamide. The involvement of the endocannabinoid system in pain and other nervous system disorders has made FAAH an attractive target for drug development. Companion molecular imaging probes are needed, however, to assess FAAH inhibition in the nervous system in vivo.

Volumetric MRI and MRS provide sensitive measures of Alzheimer's disease neuropathology in inducible Tau transgenic mice (rTg4510).

The purpose of this study was to determine if in vivo high resolution 3D MRI and localized (1)H MR spectroscopy (MRS) can detect brain findings resembling Alzheimer's disease in a transgenic mouse model of Tau pathology. Seven double transgenic rTg4510 female mice and 7 age-matched wild-type (wt) female mice were evaluated at 5 months of age. To confirm the usefulness and consistency of in vivo MRI/S, we also scanned the brains of 14 male mice (7 rTg4510 and 7 age-matched wt) at 8 months of age.

In vivo MRI assessment of knee cartilage in the medial meniscal tear model of osteoarthritis in rats.

We present a new approach for quantifying the degradation of knee cartilage in the medial meniscal tear (MMT) model of osteoarthritis in the rat. A statistical strategy was used to guide the selection of a region of interest (ROI) from the images obtained from a pilot study. We hypothesize that this strategy can be used to localize a region of cartilage most vulnerable to MMT-induced damage.

Characterizing the regional structural difference of the brain between tau transgenic (rTg4510) and wild-type mice using MRI.

rTg4510 transgenic mouse model demonstrates features resembling Alzheimer's disease including neurofibrillary degeneration and progressive neuronal loss. We investigated the volumetric differences of brain structures between transgenic and wild-type mice using MR images of fourteen 5.5 month old female mice.

In vivo micro computed tomography of subchondral bone in the rat after intra-articular administration of monosodium iodoacetate.

Osteoarthritis (OA) is a degenerative disease that is characterized by joint discomfort, loss of articular cartilage, and changes to the subchondral bone. Studies to elucidate the pathophysiology of OA have been hampered by the lack of a rapid, reproducible animal model that mimics the structural changes associated with the disease. A single intra-articular injection of mono-iodoacetate (MIA), an inhibitor of glycolysis, into the femorotibial joint of rodents promotes loss of articular cartilage similar to that noted in human OA.

Pleiotropic effects of HMG-CoA reductase inhibitors.

HMG-CoA reductase, in addition to being the rate-limiting enzyme in the cholesterol biosynthetic pathway, is involved in the regulation of receptors for low-density lipoprotein (LDL)-cholesterol. Clinical studies in men and women demonstrate that inhibitors of HMG-CoA reductase (statins), by reducing plasma cholesterol, may limit the development of atherosclerosis and reduce the risk of mortality and ischemic events. Preclinical evidence suggests that under controlled conditions of plasma cholesterol lowering, statins may have ancillary properties or pleiotropic effects, which may directly limit atherosclerosis progression.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is induced upon monocyte differentiation and is expressed in human atheroma.

Objective: Because extracellular matrix metalloproteinase inducer (EMMPRIN), a tumor cell-derived protein, induces matrix metalloproteinases (MMPs) in fibroblasts and because MMPs are important in atheroma formation, we investigated if EMMPRIN was expressed in granulocyte/macrophage-colony stimulating factor (GM-CSF)-differentiated human peripheral blood monocytes (HPBM) and macrophage foam cells. In addition, EMMPRIN was studied for its expression in human atheroma.

Methods And Results: After 10 days of GM-CSF-induced monocyte differentiation, EMMPRIN mRNA increased 5- to 8-fold relative to undifferentiated monocytes.

Characterization of the promoter of human extracellular matrix metalloproteinase inducer (EMMPRIN).

Monocyte-derived macrophages play a central role in atherosclerotic lesion formation and potentially plaque destabilization by expression of matrix metalloproteinases (MMPs); however, mechanisms associated with stimulating MMP production are not clearly understood. EMMPRIN, which is expressed by human cancer cells and macrophages, present in human, mouse and rabbit atherosclerosis and noted to induce MMPs may be involved. A DNA fragment containing 1797 bp 5' upstream of the EMMPRIN gene and the transcription start site was generated by polymerase chain reaction and cloned into a luciferase reporter gene vector, pGL3-basic.

Molecular sieving and mass spectroscopy reveal enhanced collagen degradation in rabbit atheroma.

Background: Collagen degradation is the major mechanism of atherosclerotic plaque destabilization. It is unknown whether collagen breakdown is involved into formation of early atherosclerotic lesions.

Methods: Current paper describes a novel collagen degradation assay based on a combination of molecular sieving and mass spectroscopy.

The combined effect of inhibiting both ACAT and HMG-CoA reductase may directly induce atherosclerotic lesion regression.

We hypothesized that coadministration of avasimibe and simvastatin would limit size, composition and extent of atherosclerotic lesions and potentially promote lesion regression, since bioavailable ACAT inhibitors decrease monocyte-macrophage enrichment and HMG-CoA reductase inhibitors limit smooth muscle cell migration and proliferation. Male New Zealand white rabbits were sequentially fed a 0.5% cholesterol, 3% peanut oil, 3% coconut oil diet for 9 weeks and a chow-fat diet for 6 weeks prior to drug administration.

Matrix metalloproteinase inhibition attenuates left ventricular remodeling and dysfunction in a rat model of progressive heart failure.

Background: Matrix metalloproteinase (MMP) activation contributes to tissue remodeling in several disease states, and increased MMP activity has been observed in left ventricular (LV) failure. The present study tested the hypothesis that MMP inhibition would influence LV remodeling and function in developing LV failure.

Methods And Results: LV size and function were measured in 5 groups of rats: (1) obese male spontaneously hypertensive heart failure rats (SHHF) at 9 months (n=10), (2) SHHF at 13 months (n=12), (3) SHHF rats treated with an MMP inhibitor during months 9 to 13 (PD166793 5 mg.

MMP/TIMP expression in spontaneously hypertensive heart failure rats: the effect of ACE- and MMP-inhibition.

Objective: Determine the effect of a matrix metalloproteinase inhibitor (MMPi) and angiotensin converting enzyme inhibitor (ACEi) on collagen, MMP, tissue inhibitors of MMPs (TIMPs) expression in the spontaneously hypertensive heart failure (SHHF) rat.

Methods: Six groups were tested: normotensive 9- and 13-month-old Wistar-Furth (WF) rats, 9-month-old SHHFs (compensatory hypertrophy), 13-month-old SHHFs with HF, and 13-month-old SHHFs orally administered with either an MMPi (PD166793, 5 mgkg(-1)day(-1)) or ACEi (quinapril, 10 mgkg(-1)day(-1)) for 4 months. Collagen volume fraction was assessed histomorphometrically.

The ACAT inhibitor avasimibe reduces macrophages and matrix metalloproteinase expression in atherosclerotic lesions of hypercholesterolemic rabbits.

Given the significance of cholesteryl ester (CE) accumulation in macrophage foam cell formation, we hypothesized that inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) would produce a histologically stable lesion by limiting macrophage enrichment and thereby a source of matrix metalloproteinases (MMPs). Male New Zealand White rabbits were sequentially fed a cholesterol/fat diet for 9 weeks, a fat-only diet for 6 weeks, and 25 mg/kg avasimibe for 7 to 8 weeks. Avasimibe had no effect on plasma total cholesterol exposure.