Binding Affinity, Selectivity, and Pharmacokinetics of the Oxytocin Receptor Antagonist L-368,899 in the Coyote (Canis latrans).

L-368,899 is a selective small-molecule oxytocin receptor (OXTR) antagonist originally developed in the 1990s to prevent preterm labor. Although its utility for that purpose was limited, L-368,899 is now one of the most commonly used drugs in animal research for the selective blockade of neural OXTR after peripheral delivery. A growing number of rodent and primate studies have used L-368,899 to evaluate whether certain behaviors are oxytocin dependent. These studies have improved our understanding of oxytocin's function in the brains of rodents and monkeys, but very little work has been done in other mammals, and only a single paper in macaques has provided any evidence that L-368,899 can be detected in the CNS after peripheral delivery. The current study sought to extend those findings in a novel species: coyotes ( Canis latrans ). Coyotes are ubiquitous North American canids that form long-term monogamous pair-bonds. Although monogamy is rare in rodents and primates, all wild canid species studied to date exhibit social monogamy. Coyotes are therefore an excellent model organism for the study of oxytocin and social bonds. Our goal was to determine whether L-368,899 is a viable candidate for future use in behavioral studies in coyotes. We used captive coyotes at the USDA National Wildlife Research Center's Predator Research Facility to evaluate the pharmacokinetics of L-368,899 in blood and CSF during a 90-min time course after intramuscular injection. We then characterized the binding affinity and selectivity of L-368,899 to coyote OXTR and the structurally similar vasopressin 1a receptor. We found that L-368,899 peaked in CSF at 15 to 30 min after intramuscular injection and slowly accumulated in blood. L-368,899 was 40 times more selective for OXTR than vasopressin 1a receptors and bound to the coyote OXTR with an affinity of 12 nM. These features of L-368,899 support its utility in future studies to probe the oxytocin system of coyotes.

Spontaneous Atrial Fibrillation in Transgenic Goats With TGF (Transforming Growth Factor)-ß1 Induced Atrial Myopathy With Endurance Exercise.

BACKGROUND: There is increasing evidence that endurance exercise is associated with increased risk of atrial fibrillation (AF). However, it is unknown if the relationship between endurance exercise and AF is dependent on an atrial myopathy. METHODS: Six cardiac-specific TGF (transforming growth factor)-ß1 transgenic and 6 wild-type (WT) goats were utilized for these studies. Pacemakers were implanted in all animals for continuous arrhythmia monitoring and AF inducibility. AF inducibility was evaluated using 5 separate 10 s bursts of atrial pacing (160-200 ms). Three months of progressive endurance exercise (up to 90 minutes at 4.5 mph) was performed. Quantitative assessment of circulating microRNAs and inflammatory biomarkers was performed. RESULTS: Sustained AF (≥30 s) was induced with 10 s of atrial pacing in 4 out of 6 transgenic goats compared with 0 out of 6 WT controls at baseline (P<0.05). No spontaneous AF was observed at baseline. Interestingly, between 2 and 3 months of exercise 3 out of 6 transgenic animals developed self-terminating spontaneous AF compared with 0 out of 6 WT animals (P<0.05). There was an increase in AF inducibility in both transgenic and WT animals during the first 2 months of exercise with partial normalization at 3 months (transgenic 67%; 100%; 83% versus WT 0%; 67%; 17%). These changes in AF susceptibility were associated with a decrease in circulating microRNA-21 and microRNA-29 during the first 2 months of exercise with partial normalization at 3 months in both transgenic and WT animals. Finally, MMP9 (matrix metallopeptidase 9) was increased during the second and third months of exercise training. CONCLUSIONS: This study demonstrates a novel transgenic goat model of cardiac fibrosis (TGF-ß1 overexpression) to demonstrate that endurance exercise in the setting of an underlying atrial myopathy increases the incidence of spontaneous AF. Furthermore, endurance exercise seems to increase inducible AF secondary to altered expression of key profibrotic biomarkers that is independent of the presence of an atrial myopathy.

Comparison of catheter thrombogenicity in a modified chandler loop model using goat blood.

Thrombogenicity testing is a key component in the development of medical devices intended for contact with blood. The Chandler loop system has previously been used as an in vitro thrombogenicity testing method. In this study, we used a modified version of the Chandler loop model to evaluate its capacity to detect differential thrombogenic potential of different catheter materials using goat blood. We also sought to determine the optimal experimental conditions for detecting the thrombogenicity of catheter material. Using the Chandler loop system with goat blood we demonstrated that silicone catheters had a stronger thrombogenicity as compared to polyurethane catheters as evidenced by significantly larger thrombi (p < 0.001) and higher infusion pressures (p < 0.05). This is consistent with many, but not all, previous in vitro and in vivo studies comparing polyurethane to silicone catheters. The use of this modified Chandler loop system with goat blood may provide an additional in vitro testing platform for thrombogenicity testing of catheters. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3143-3151, 2018.

Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection.

Zika virus (ZIKV) causes no-to-mild symptoms or severe neurological disorders. To investigate the importance of viral and host genetic variations in determining ZIKV infection outcomes, we created three full-length infectious cDNA clones as bacterial artificial chromosomes for each of three spatiotemporally distinct and genetically divergent ZIKVs: MR-766 (Uganda, 1947), P6-740 (Malaysia, 1966), and PRVABC-59 (Puerto Rico, 2015). Using the three molecularly cloned ZIKVs, together with 13 ZIKV region-specific polyclonal antibodies covering nearly the entire viral protein-coding region, we made three conceptual advances: (i) We created a comprehensive genome-wide portrait of ZIKV gene products and their related species, with several previously undescribed gene products identified in the case of all three molecularly cloned ZIKVs. (ii) We found that ZIKV has a broad cell tropism in vitro, being capable of establishing productive infection in 16 of 17 animal cell lines from 12 different species, although its growth kinetics varied depending on both the specific virus strain and host cell line. More importantly, we identified one ZIKV-non-susceptible bovine cell line that has a block in viral entry but fully supports the subsequent post-entry steps. (iii) We showed that in mice, the three molecularly cloned ZIKVs differ in their neuropathogenicity, depending on the particular combination of viral and host genetic backgrounds, as well as in the presence or absence of type I/II interferon signaling. Overall, our findings demonstrate the impact of viral and host genetic variations on the replication kinetics and neuropathogenicity of ZIKV and provide multiple avenues for developing and testing medical countermeasures against ZIKV.

The Influence of Oblique Angle Forced Exercise in Surgically Destabilized Stifle Joints Is Synergistic with Bone, but Antagonistic with Cartilage in an Ovine Model of Osteoarthritis.

Large animal models of osteoarthritis are a necessary testing ground for FDA approval of human medicine applications. Sheep models have advantages over other available large animals, but development and progression of osteoarthritis in sheep is exceedingly slow, which handicaps progress in development of potential treatments. We combined oblique angle forced exercise to increase stress on the stifle, with surgical destabilization to hasten the development of osteoarthritis in ewes. Methods for early detection of clinical signs included radiography, urine, and serum biomarker assays and gait analysis and ex vivo we used microcomputed tomography and macroscopic joint analysis. Our model was able to produce clinically detectable signs of osteoarthritis in a relatively short period (14 weeks). Changes in bone were highly correlated between microcomputed tomography and radiographic analysis and changes in cartilage correlated well between urinary glycosaminoglycan levels and serum aggrecanase analyses. Exercise improved the negative effects of destabilization in bone but exacerbated the negative effects of destabilization in cartilage. These observations suggest that we may need to consider treatments for bone and cartilage separately. These results represent an improved large animal model of osteoarthritis with rapid onset of disease and superior detection of bone and soft tissue changes.

Increased Susceptibility to Atrial Fibrillation Secondary to Atrial Fibrosis in Transgenic Goats Expressing Transforming Growth Factor-ß1.

INTRODUCTION: Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF-ß1 and investigated the changes in the cardiac structure and function leading to AF. METHODS AND RESULTS: Transgenic goats with cardiac specific overexpression of constitutively active TGF-ß1 were generated by somatic cell nuclear transfer. We examined myocardial tissue, ECGs, echocardiographic data, and AF susceptibility in transgenic and wild-type control goats. Transgenic goats exhibited significant increase in fibrosis and myocyte diameters in the atria compared to controls, but not in the ventricles. P-wave duration was significantly greater in transgenic animals starting at 12 months of age, but no significant chamber enlargement was detected, suggesting conduction slowing in the atria. Furthermore, this transgenic goat model exhibited a significant increase in AF vulnerability. Six of 8 transgenic goats (75%) were susceptible to AF induction and exhibited sustained AF (>2 minutes), whereas none of 6 controls displayed sustained AF (P < 0.01). Length of induced AF episodes was also significantly greater in the transgenic group compared to controls (687 ± 212.02 seconds vs. 2.50 ± 0.88 seconds, P < 0.0001), but no persistent or permanent AF was observed. CONCLUSION: A novel transgenic goat model with a substrate for AF was generated. In this model, cardiac overexpression of TGF-ß1 led to an increase in fibrosis and myocyte size in the atria, and to progressive P-wave prolongation. We suggest that these factors underlie increased AF susceptibility.

Diagnostic imaging and pacemaker implantation in a domestic goat with persistent left cranial vena cava.

Difficulty was encountered with the insertion of a right atrial pacing lead via the left jugular vein during lead and pacemaker implantation in a clinically normal goat as part of an ongoing rapid atrial pacing - induced atrial fibrillation research project. Fluoroscopic visualization of an abnormal lead advancement path prompted angiographic assessment which revealed a persistent left cranial vena cava (PLCVC) and prominent coronary sinus communicating with the right atrium. Angiography facilitated successful advancement and securing of the pacing lead into the right side of the interatrial septum. Cardiac magnetic resonance imaging/magnetic resonance angiography (MRI/MRA) allowed further characterization of this rare venous anomaly. Even though PLCVC has been reported once in a goat, to the authors' knowledge this is the first report to include MRI/MRA characterization of PLCVC and prominent coronary sinus with successful cardiac pacemaker implantation using the PLCVC.

Potent in vitro activity of the albumin fusion type 1 interferons (albumin-interferon-alpha and albumin-interferon-beta) against RNA viral agents of bioterrorism and the severe acute respiratory syndrome (SARS) virus.

BACKGROUND: The type 1 interferons (INF-alpha and INF-beta) are potent antiviral agents. Albumin-INF-alpha and albumin-INF-beta are novel recombinant proteins consisting of IFN-alpha or IFN-beta genetically fused to human albumin. METHODS: The in vitro antiviral activity of albumin-IFN-alpha was evaluated against representative bioterrorism viral agents and the severe acute respiratory syndrome virus. Antiviral activity was assessed using inhibition of cytopathic effect and neutral red staining. RESULTS: EC(50) values for albumin-IFN-alpha ranged from <0.1 ng/ml for Punta Toro virus to 65 ng/ml for Venezuelan equine encephalitis virus in the neutral red assay. Albumin-IFN-beta showed 75- and 360-fold greater in vitro activity than albumin-IFN-alpha against Ebola virus and severe acute respiratory syndrome, respectively. CONCLUSION: Further evaluation of these long-acting albumin-IFN fusion proteins as prophylactic or therapeutic agents against these viral agents of bioterrorism in relevant primate models is warranted.

West Nile virus-induced acute flaccid paralysis is prevented by monoclonal antibody treatment when administered after infection of spinal cord neurons.

Acute flaccid polio-like paralysis occurs during natural West Nile virus (WNV) infection in a subset of cases in animals and humans. To evaluate the pathology and the possibility for therapeutic intervention, the authors developed a model of acute flaccid paralysis by injecting WNV directly into the sciatic nerve or spinal cord of hamsters. By directly injecting selected sites of the nervous system with WNV, the authors mapped the lesions responsible for hind limb paralysis to the lumbar spinal cord. Immunohistochemical analysis of spinal cord sections from paralyzed hamsters revealed that WNV-infected neurons localized primarily to the ventral motor horn of the gray matter, consistent with the polio-like clinical presentation. Neuronal apoptosis and diminished cell function were identified by TUNEL (terminal deoxynucleotidyl transferase-mediated BrdUTP nick end labeling) and choline acetyltransferase staining, respectively. Administration of hE16, a potently neutralizing humanized anti-WNV monoclonal antibody, 2 to 3 days after direct WNV infection of the spinal cord, significantly reduced paralysis and mortality. Additionally, a single injection of hE16 as late as 5 days after WNV inoculation of the sciatic nerve also prevented paralysis. Overall, these experiments establish that WNV-induced acute flaccid paralysis in hamsters is due to neuronal infection and injury in the lumbar spinal cord and that treatment with a therapeutic antibody prevents paralysis when administered after WNV infection of spinal cord neurons.

Increased blood-brain barrier permeability is not a primary determinant for lethality of West Nile virus infection in rodents.

Blood-brain barrier (BBB) permeability was evaluated in mice and hamsters infected with West Nile virus (WNV, flavivirus) as compared to those infected with Semliki Forest (alphavirus) and Banzi (flavivirus) viruses. BBB permeability was determined by measurement of fluorescence in brain homogenates or cerebrospinal fluid (CSF) after intraperitoneal (i.p.) injection of sodium fluorescein, by macroscopic examination of brains after i.p. injection of Evans blue, or by measurement of total protein in CSF compared to serum. Lethal infection of BALB/c mice with Semliki Forest virus and Banzi virus caused the brain : serum fluorescence ratios to increase from a baseline of 2-4% to as high as 11 and 15%, respectively. Lethal infection of BALB/c mice with WNV did not increase BBB permeability. When C57BL/6 mice were used, BBB permeability was increased in some, but not all, of the WNV-infected animals. A procedure was developed to measure BBB permeability in live WNV-infected hamsters by comparing the fluorescence in the CSF, aspirated from the cisterna magnum, with the fluorescence in the serum. Despite a time-dependent tendency towards increased BBB permeability in some WNV-infected hamsters, the highest BBB permeability values did not correlate with mortality. These data indicated that a measurable increase in BBB permeability was not a primary determinant for lethality of WNV infection in rodents. The lack of a consistent increase in BBB permeability in WNV-infected rodents has implications for the understanding of viral entry, viral pathogenesis and accessibility of the CNS of rodents to drugs or effector molecules.

Defining limits of treatment with humanized neutralizing monoclonal antibody for West Nile virus neurological infection in a hamster model.

A potent anti-West Nile virus (anti-WNV)-neutralizing humanized monoclonal antibody, hE16, was previously shown to improve the survival of WNV-infected hamsters when it was administered intraperitoneally (i.p.), even after the virus had infected neurons in the brain. In this study, we evaluated the therapeutic limit of hE16 for the treatment of WNV infection in hamsters by comparing single-dose peripheral (i.p.) therapy with direct administration into the pons through a convection-enhanced delivery (CED) system. At day 5 after infection, treatments with hE16 by the peripheral and the CED routes were equally effective at reducing morbidity and mortality. In contrast, at day 6 only the treatment by the CED route protected the hamsters from lethal infection. These experiments suggest that hE16 can directly control WNV infection in the central nervous system. In support of this, hE16 administered i.p. was detected in a time-dependent manner in the serum, cerebrospinal fluid (CSF), cerebral cortex, brain stem, and spinal cord in CSF. A linear relationship between the hE16 dose and the concentration in serum was observed, and maximal therapeutic activity occurred at doses of 0.32 mg/kg of body weight or higher, which produced serum hE16 concentrations of 1.3 microg/ml or higher. Overall, these data suggest that in hamsters hE16 can ameliorate neurological disease after significant viral replication has occurred, although there is a time window that limits therapeutic efficacy.

Prophylactic treatment with recombinant Eimeria protein, alone or in combination with an agonist cocktail, protects mice from Banzi virus infection.

A recombinant Eimeria protozoan protein antigen (rEA) has been shown to have antitumor and antiviral activity. The purpose of this study was to determine the effect of rEA treatment alone or in combination with an agonist cocktail consisting of granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-gamma), interleukin 4 (IL-4), and anti CD-40 antibody, in the treatment of Banzi virus (BV) disease in BALB/c mice. Treatment with rEA resulted in a significant increase in survival, weight gain, and mean day to death in BV-infected mice and resulted in a significant decrease in brain virus titer. Treatment with rEA, in combination with a 4-agonist cocktail, improved disease parameters to a greater degree than rEA treatment alone. The effect of treatment with a reduced concentration of agonist cocktail or fewer components of the agonist cocktail, in combination with rEA, on disease outcome in BV-infected mice was also investigated. Treatment with rEA, alone or in combination with agonist cocktail, 24h after virus challenge did not improve disease. Treatment with rEA, alone or in combination with an agonist cocktail, is efficacious for the prophylaxis of BV infection in mice.

Correlation between breakdown of the blood-brain barrier and disease outcome of viral encephalitis in mice.

Changes in the permeability of the blood-brain barrier (BBB) were evaluated in two mouse models of viral encephalitis. The ability of sodium fluorescein (NaFl) to cross the BBB from the serum into the central nervous system was assayed in animals inoculated with virulent strains of either Banzi or Semliki Forest viruses. To test the hypothesis that increases in BBB permeability were associated with poor disease outcome subsequent experiments measured BBB permeability in conjunction with treatment with the interferon inducer Ampligen (poly I:poly C(12)U). A single intraperitoneal injection of Ampligen (1 mg/kg) administered either 24 h or 4-6 h before, but not 24 h after, virus inoculation with Banzi virus provided significant improvements in survival, viral brain titers, weight change and BBB permeability. In comparison, a similar treatment with Ampligen administered either 24 h or 4-6 h before inoculation with Semliki Forest virus was able to significantly improve weight change, and BBB permeability, but only animals receiving Ampligen 4-6 h pre-virus showed a significantly improved mortality. In general, it was found that evaluation of BBB permeability was a more sensitive indicator of disease outcome and the antiviral efficacy Ampligen than either weight change or brain viral titers.

Humanized monoclonal antibody against West Nile virus envelope protein administered after neuronal infection protects against lethal encephalitis in hamsters.

Humans infected with West Nile virus (WNV) may clinically present with symptoms that are suggestive of neurological infection. Nearly all treatments of WNV disease have been effective in animal models only if administered before or soon after viral challenge. Here, we evaluated whether a potent neutralizing anti-WNV humanized monoclonal antibody (MAb), hE16, could improve the course of disease in a hamster model when administered after the virus had infected neurons in the brain. Five days after viral injection, WNV was detected in the brains of hamsters by cytopathic assay, quantitative reverse-transcription polymerase chain reaction, and immunohistochemical staining of WNV envelope in neurons. Notably, 80%-90% of the hamsters treated 5 days after viral injection by intraperitoneal injection with hE16 survived the disease, compared with 37% of the placebo-treated hamsters (P< or =.001). The hamsters that received hE16 directly in the brain also exhibited markedly improved survival rates, compared with those in the placebo-treated hamsters. In prospective experiments, hamsters with high levels of infectious WNV in their cerebrospinal fluid were also protected by hE16 when administered 5 days after viral injection. These experiments suggest that humanized MAbs with potent neutralizing activity are a possible treatment for human patients after WNV has infected neurons in the central nervous system.

Treatment of West Nile virus-infected mice with reactive immunoglobulin reduces fetal titers and increases dam survival.

The objectives of this study were to determine if injection of West Nile virus (WNV) into timed-pregnant mice would result in fetal infection and if administration of WNV-reactive immunoglobulin would increase dam survival and reduce fetal viral titers. Dams injected on 7.5 days post-coitus (dpc) had detectable viral titers in the placenta 10.5dpc with a mean titer of 10(4.9) 50% cell-culture infectious doses per gram of tissue (CCID(50)/g tissue). The mean placental titer increased to 10(8.6)CCID(50)/g tissue at 12.5dpc. Infectious virus was detectable 12.5dpc in 10 of 10 fetuses with a mean titer of 10(7.5)CCID(50)/g tissue. Treatment of dams (challenged with WNV on 7.5dpc) with WNV-reactive human immunoglobulin (Ig) on 8.5 and 9.5dpc resulted in a significant reduction of virus in fetuses as compared with non-reactive human Ig-treated females on 12.5dpc (P< or =0.001). Treatment also resulted in survival of dams to term. Treatment of dams with WNV-reactive human Ig on 12.5 and 13.5dpc also resulted in reduction of viral titer on 14.5dpc, indicating that later treatment may also be efficacious. This suggests that Ig treatment may be useful in treating fetal WNV infection in women.

Modeling hamsters for evaluating West Nile virus therapies.

A hamster model infected with a New York crow brain isolate of West Nile virus (WNV) was characterized for evaluating potential antiviral therapies. Older hamsters (7-11 weeks old) had a lower mortality of approximately 50% and more apparent disease signs as compared to >90% mortality in younger hamsters and mice. Disease signs such as limb strength, lacrimation, front limb tremors, somnolence, and deficiencies in neurological responses were noted at different times after viral injection. Weight loss was a marker for WNV disease signs, whereas, the ability to climb up an inclined ramp was associated with whether the animals survived the disease or died. Infectious WNV assays performed on tissues from hamsters during development of the infection indicated that viral titers peaked first in plasma, but that titers were eventually highest in kidney tissue. Viral titers achieved maximal levels in brain tissue on 6 dpi, which was 1-2 days before strong neurological signs and death started to occur. Maximal spleen and plasma titers were achieved sooner in young hamsters as compared with older hamsters, which correlated with increased susceptibility. To test the hypothesis that older hamsters would be more sensitive for identifying antiviral effects, Infergen, a consensus human interferon-alpha highly active against WNV in cell culture, was administered subcutaneously to older and younger hamsters beginning on 2 dpi. The effects of Infergen on weight change, survival, and climbing ability of infected animals were more apparent in older hamsters than in younger hamsters. The use of older hamsters is another WNV-infectious model, in addition to mice, for evaluating potential antiviral therapies.