Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

Development of chlorine-induced lung injury in the anesthetized, spontaneously breathing pig.

Chlorine is a toxic industrial chemical produced in vast quantities globally, being used in a range of applications such as water purification, sanitation and industrial processes. Its use and transport cannot be restricted; exposure may occur following accidental or deliberate releases. The OPCW recently verified the use of chlorine gas against civilians in both Syria and Iraq. Chlorine inhalation produces damage to the lungs, which may result in the development of an acute lung injury, respiratory failure and death. Treatment remains an intractable problem. Our objective was to develop a clinically relevant pre-clinical model of a moderate to severe lung injury in the pig. This would enable future assessment of therapeutic drugs or interventions to be implemented in the pre-hospital phase after exposure. Due to the irritant nature of chlorine, a number of strategies for exposing terminally anesthetized pigs needed to be investigated. A number of challenges (inconsistent acute changes in respiratory parameters; early deaths), resulted in a moderate to severe lung injury not being achieved. However, most pigs developed a mild lung injury by 12 h. Further investigation is required to optimize the model and enable the assessment of therapeutic candidates. In this paper we describe the exposure strategies used and discuss the challenges encountered in establishing a model of chlorine-induced lung injury. A key aim is to assist researchers navigating the challenges of producing a clinically relevant model of higher dose chlorine exposure where animal welfare is protected by use of terminal anesthesia.

The Experimental Design Assistant.

Addressing the common problems that researchers encounter when designing and analysing animal experiments will improve the reliability of in vivo research. In this article, the Experimental Design Assistant (EDA) is introduced. The EDA is a web-based tool that guides the in vivo researcher through the experimental design and analysis process, providing automated feedback on the proposed design and generating a graphical summary that aids communication with colleagues, funders, regulatory authorities, and the wider scientific community. It will have an important role in addressing causes of irreproducibility.

Bioavailability of subcutaneous and intramuscular administrated buprenorphine in New Zealand White rabbits.

BACKGROUND: Buprenorphine is one of the most used analgesics for postoperative pain in rabbits. The recommended dose in rabbits (0.01-0.05 mg/kg) is the same for intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration, despite lack of pharmacokinetic data. Five male and five female New Zealand White rabbits (mean ± SD body weight 3.1 ± 0.3 kg) were administered 0.05 mg/kg buprenorphine by the IV, IM and SC routes and 0.1 mg/kg by the SC route, in a cross-over design with two-week wash-out periods between treatments. Blood was collected before, and up to 8 h post buprenorphine injection, for determination of serum levels by UPHLC-MS/MS. RESULTS: The area under the time concentration curve (AUC0-t) was lower after SC (398 ± 155 ng/mL/min) than IM (696 ± 168 ng/mL/min, p < 0.001) and IV (789 ± 189 ng/mL/min, p < 0.001) administration. The maximum serum concentration was lower after SC (2.2 ± 1.4 ng/mL) than after IM (11 ± 3.2 ng/mL) administration (p < 0.001). The bioavailability was lower after SC (50 ± 19%) than after IM (95 ± 21%) administration (p = 0.006). The elimination half-life was longer after SC (260 ± 120 min) than after IM (148 ± 26 min, p = 0.002) as well as IV (139 ± 33 min) injection (p < 0.001). An increase in the SC dose from 0.05 to 0.1 mg/kg resulted in an increase in the area under the time concentration curve of 50% in female (p = 0.022) and 165% in male rabbits (p < 0.001). The bioavailability did not change in the females (36 ± 14%, p = 0.6), whereas it increased in the males (71 ± 23%, p = 0.008). CONCLUSIONS: The lower bioavailability of 0.05 mg/kg buprenorphine after SC administration could explain the lack of efficacy seen in clinical pain studies in rabbits, using this route. For immediate pain relief, IV or IM administration is therefore be recommended, whereas SC administration may be useful to sustain analgesic serum levels, once efficient pain relief has been achieved. The current data do not support an increase in dose to compensate for the lower SC bioavailability.

When neuroscience met clinical pathology: partitioning experimental variation to aid data interpretation in neuroscience.

In animal experiments, neuroscientists typically assess the effectiveness of interventions by comparing the average response of groups of treated and untreated animals. While providing useful insights, focusing only on group effects risks overemphasis of small, statistically significant but physiologically unimportant, differences. Such differences can be created by analytical variability or physiological within-individual variation, especially if the number of animals in each group is small enough that one or two outlier values can have considerable impact on the summary measures for the group. Physicians face a similar dilemma when comparing two results from the same patient. To determine whether the change between two values reflects disease progression or known analytical and physiological variation, the magnitude of the difference between two results is compared to the reference change value. These values are generated by quantifying analytical and within-individual variation, and differences between two results from the same patient are considered clinically meaningful only if they exceed the combined effect of these two sources of 'noise'. In this article, we describe how the reference change interval can be applied within neuroscience. This form of analysis provides a measure of outcome at an individual level that complements traditional group-level comparisons, and therefore, introduction of this technique into neuroscience can enrich interpretation of experimental data. It can also safeguard against some of the possible misinterpretations that may occur during analysis of the small experimental groups that are common in neuroscience and, by illuminating analytical error, may aid in design of more efficient experimental methods.

Characterization of the novel positive allosteric modulator of the metabotropic glutamate receptor 4 ADX88178 in rodent models of neuropsychiatric disorders.

There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis.

Assessment of the protective effect of Imvamune and Acam2000 vaccines against aerosolized monkeypox virus in cynomolgus macaques.

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference (P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.

Angiopoietin-1 regulates microvascular reactivity and protects the microcirculation during acute endothelial dysfunction: role of eNOS and VE-cadherin.

The growth factor angiopoietin-1 (Ang-1) plays an essential role in angiogenesis and vascular homeostasis. Nevertheless, the role of Ang-1 in regulating vascular tone and blood flow is largely unexplored. Endothelial nitric oxide synthase (eNOS) and the junctional protein VE-cadherin are part of the complex signalling cascade initiated by Ang-1 in endothelial cells. In this study, we aimed to investigate the mechanisms underlying acute effects of Ang-1 on microvascular reactivity, permeability and blood flow, and hypothesise that eNOS and VE-cadherin underpin Ang-1 mediated vascular effects that are independent of angiogenesis and proliferation. Myography of isolated microarterioles from male C3H/HeN mice (7-10 weeks) was employed to measure vascular reactivity in vitro. Microcirculatory function in vivo was evaluated by intravital microscopy and Doppler fluximetry in dorsal window chambers. Ang-1 and its stable variant MAT.Ang-1 induced a concentration-dependent vasodilation of arterioles in vitro, which was blocked with nitric oxide (NO) synthesis inhibitor l-NAME. In vivo, MAT.Ang-1 restored to control levels l-NAME induced peripheral vasoconstriction, decreased blood flow and microvascular hyperpermeability. Tissue protein expression of VE-cadherin was reduced by NOS inhibition and restored to control levels by MAT.Ang-1, whilst VE-cadherin phosphorylation was increased by l-NAME and subsequently reduced by MAT.Ang-1 administration. Moreover, MAT.Ang-1 alone did not modulate systemic levels of angiogenetic factors. Our novel findings report that Ang-1 induces arteriolar vasodilation via release of NO, suggesting that Ang-1 is an important regulator of microvascular tone. As MAT.Ang-1 ameliorates detrimental effects on the microcirculation induced by inhibition of NO synthesis and stabilizes the endothelial barrier function through VE-cadherin, we propose that this Ang-1 variant may serve as a novel therapeutic agent to protect the microcirculation against endothelial dysfunction.

Review of the statistical analysis of the dog telemetry study.

In 2008, the PSI Toxicology Special Interest Group met to discuss the design and analysis of dog telemetry studies. The dog telemetry study is one component of the integrated cardiovascular assessment required by regulatory bodies. Although there are guidelines for these studies, little is said about the statistical analysis. With parameters of interest measured continually over time, in studies typically involving four dogs, the analysis is not straightforward. This has led to many different types of analysis being proposed in the literature, with many different methods applied within the pharmaceutical industry itself. This paper summarizes the PSI Toxicology group's discussions and recommendations around these issues.

Recommendations on the statistical analysis of the Comet assay.

In 2010, the Statisticians in the Pharmaceutical Industry (PSI) Toxicology Special Interest Group met to discuss the design and analysis of the Comet assay. The Comet assay is one potential component of the package of safety studies required by regulatory bodies. As these studies usually involve a three-way nested experimental design and as the distribution of the measured response is usually either lognormal or lognormal plus a point mass at zero, the analysis is not straightforward. This has led to many different types of analysis being proposed in the literature, with several different methods applied within the pharmaceutical industry itself. This article summarises the PSI Toxicology Group's discussions and recommendations around these issues.

An assessment of the statistical methods used to analyse toxicology studies.

The Statisticians in the Pharmaceutical Industry Toxicology Special Interest Group has collated and compared statistical analysis methods for a number of toxicology study types including general toxicology, genetic toxicology, safety pharmacology and carcinogenicity. In this paper, we present the study design, experimental units and analysis methods.

Sampling optimization for blend monitoring of a low dose formulation in a tablet press feed frame using spatially resolved near-infrared spectroscopy.

In-line measurements of low dose blends in the feed frame of a tablet press were performed for API concentration levels as low as 0.10% w/w. The proposed methodology utilizes the advanced sampling capabilities of a Spatially Resolved Near-Infrared (SR-NIR) probe to develop Partial Least-Squares calibration models. The fast acquisition speed of multipoint spectra allowed the evaluation of different numbers of co-adds and feed frame paddle speeds to establish the optimum conditions of data collection to predict low potency blends. The interaction of the feed frame paddles with the SR-NIR probe was captured with high resolution and allowed the implementation of a spectral data selection criterion to remove the effect of the paddles from the calibration and testing process. The method demonstrated accuracy and robustness when predicting drug concentrations across different feed frame paddle speeds.

Potentiation of the anticonvulsant efficacy of sodium channel inhibitors by an NK1-receptor antagonist in the rat.

PURPOSE: Many patients with epilepsy are refractory to anticonvulsant drugs or do not tolerate side effects associated with the high doses required to fully prevent seizures. Antagonists of neurokinin-1 (NK1) receptors have the potential to reduce seizure severity, although this potential has not been fully explored in animals or humans. The present study was designed to evaluate the efficacy of the NK1-receptor antagonist, vofopitant, alone and in combination with different anticonvulsant drugs. METHODS: Studies were conducted in rats using a model of generalized seizure induced by electroshock. Drug concentrations in blood and brain were determined in parallel to distinguish pharmacodynamic from pharmacokinetic interactions. RESULTS: The NK1-receptor antagonist, GR205171 (vofopitant) had no anticonvulsant efficacy by itself, but could potentiate the anticonvulsant efficacy of lamotrigine and other sodium channel blockers. However, GR205171 had no effect on the anticonvulsant potency of either valproate or gabapentin. GR205171 did not produce central nervous system (CNS) side effects at the doses tested, and it did not potentiate side effects induced by high doses of lamotrigine. The NK1-receptor inactive enantiomer of GR205171, GR226206 did not potentiate the efficacy of lamotrigine, suggesting that effects observed with GR205171 were mediated by NK1 receptors. Analysis of the dose-effect relationship for GR205171 indicated that a high (>99%) occupancy of NK1 receptors is required for effect, consistent with previous behavioral and human clinical studies with this pharmacologic class. DISCUSSION: These results suggest that there may be benefit in adding treatment with a suitable NK1-receptor antagonist to treatment with a sodium channel blocker in patients with refractory epilepsy.

Analysis of serial magnetic resonance images of mouse brains using image registration.

The aim of this paper is to investigate techniques that can identify and quantify cross-sectional differences and longitudinal changes in vivo from magnetic resonance images of murine models of brain disease. Two different approaches have been compared. The first approach is a segmentation-based approach: Each subject at each time point is automatically segmented into a number of anatomical structures using atlas-based segmentation. This allows cross-sectional and longitudinal analyses of group differences on a structure-by-structure basis. The second approach is a deformation-based approach: Longitudinal changes are quantified by the registration of each subject's follow-up images to that subject's baseline image. In addition the baseline images can be registered to an atlas allowing voxel-wise analysis of cross-sectional differences between groups. Both approaches have been tested on two groups of mice: A transgenic model of Alzheimer's disease and a wild-type background strain, using serial imaging performed over the age range from 6-14 months. We show that both approaches are able to identify longitudinal and cross-sectional differences. However, atlas-based segmentation suffers from the inability to detect differences across populations and across time in regions which are much smaller than the anatomical regions. In contrast to this, the deformation-based approach can detect statistically significant differences in highly localized areas.

Assessing the safety and suitability of nesting material for singly housed mice with surgically fitted head plates.

Nesting material, for example shredded paper, is a common form of enrichment for laboratory mice. However, there has been limited research performed regarding its apparent safety when given to mice fitted with exteriorised devices such as head plates. Anecdotally, shredded paper has been deemed unsafe for use with such animals due to frequently observed entanglement. This study assessed the safety of four nesting materials (Pure Comfort White, Rodent Roll, Short Paper Shavings and Facial Tissue) to identify a suitable alternative to shredded paper. The four nesting materials were each tested on 5 head plate mice over a 14-day period. The quality of the nests produced was scored throughout the trial period and incidences of tangling monitored. Tangling was only observed in the Facial Tissue group, and the highest quality nest scores were recorded in the Pure Comfort White group. As shredded paper has been anecdotally alleged to cause entanglement, Pure Comfort White, Rodent Roll and Short Paper Shavings may present more suitable options, given that their use did not result in any incidences of tangling throughout this trial. Pure Comfort White was concluded to be the safest and most suitable nesting material for head plate mice as it produced nests of high quality while reducing the risk of entanglement.

Differences in monocyte: lymphocyte ratio and Tuberculosis disease progression in genetically distinct populations of macaques.

Monocyte:lymphocyte ratio (M:L) has been identified as a risk factor in development of TB disease in children and those undergoing treatment for HIV in co-infected individuals. Retrospective analysis was performed using M:L data collected from TB modelling studies performed in Rhesus macaques of Indian genotype (RM), cynomolgus macaque of Chinese genotype (CCM) and cynomolgus macaque of Mauritian genotype (MCM), which found that the more susceptible populations (RM and MCM) had higher M:L ratios than the least susceptible population (CCM). Following Mycobacterium tuberculosis exposure, significant increases in M:L ratio were observed in susceptible RM and MCM within 12 weeks of TB infection, whereas M:L in CCM remained stable, suggesting that changes in M:L ratio may also act as a biomarker of TB disease progression. The frequency of PPD-specific interferon gamma (IFNγ) secreting cells (SFU) were compared, with the more susceptible macaque populations showing an association between M:L and IFNγ SFU frequency. Investigation of the genes associated with monocyte-derived antigen presenting cells revealed differences between RM and CCM, highlighting differences in their monocyte populations, as well as overall M:L ratio. Differences in M:L ratio between macaque populations could be used to explore immunological mechanisms in susceptible populations that would complement human population studies.

Selective dopamine D4 receptor agonist (A-412997) improves cognitive performance and stimulates motor activity without influencing reward-related behaviour in rat.

Current therapies for attention deficit hyperactivity disorder comprise psychostimulants, which block the dopamine transporter and/or stimulate the release of dopamine, leading to a global elevation in extrasynaptic dopamine. These drugs are, however, associated with a series of unwanted side effects such as insomnia, anorexia, headache, stomach problems and potential drug abuse. Recent evidence suggests that the dopamine D4 receptor may represent a selective dopamine target that could mediate cognitive as well as striatal motor processes. In this study we compare the effects of a selective D4 receptor agonist, A-412997, with methylphenidate or amphetamine in preclinical models of efficacy versus abuse liability. Both methylphenidate and A-412997 improved a temporally induced deficit in the rat novel object recognition task at doses 10-fold lower than those stimulating activity. In both cases, procognitive doses were associated with elevated extracellular levels of dopamine and acetylcholine in the medial prefrontal cortex. In contrast to amphetamine, A-412997 did not mediate reward-related behaviour in the conditioned place preference paradigm, a preclinical rodent test used to assess potential abuse liability. Collectively, these data suggest that selective activation of the D4 receptor may represent a target for the treatment of attention deficit hyperactivity disorder without the potential drug abuse liability associated with current psychostimulant therapies.

Influence of intertrial interval on basal and drug-induced impulsive action in the 5-choice serial reaction time task: Effects of d-amphetamine and (±)-2,5-dimethoxy-4-iodoamphetamine (DOI).

Impulsivity is a characteristic of a number of neuropsychiatric disorders such as attention-deficit/hyperactivity disorder. The 5-choice serial reaction time task (5-CSRTT) is a rodent paradigm extensively used to assess attention and impulsivity. Notably, 5-CSRTT studies do not typically account for the reduction in premature responding, the measure of impulsive action, occurring upon repeated exposure to test sessions with long or variable intertrial intervals (ITIs). This present 5-CSRTT study investigated the use of variable ITIs (5, 10 or 15s) across 15 test days (4 training days followed by 1 drug test day per week for three weeks) as previous experience had shown that 4 training days would be sufficient to induce consistent premature response levels in male C57BL/6J mice. Once a steady state was achieved, the effects of dextroamphetamine (AMPH) and (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) were then assessed using a Latin-square design to determine whether pharmacological-induced impulsive actions depended on ITI length. Mice habituated to the variable ITI schedule after only 3days and showed consistently lower premature response levels until the end of the study. AMPH (p<0.05) and DOI (p<0.05) increased the percentage of premature responses at 15s ITI trials, while only DOI (p<0.05) increased impulsive action at 10s ITI trials. Additionally, DOI increased omission rates (p<0.001), mean correct latency (p<0.01), reward collection latency (p<0.001), and reduced the total attempted trials (p<0.001). In summary, we demonstrated that mice habituate to the variable ITI schedule, suggesting that using the variable ITI schedule during training allowed premature response rates to stabilize before commencing pharmacological testing. Moreover, in these habituated mice AMPH and DOI significantly enhanced impulsive action at the long ITI trials only. We propose that experimental design considerations can improve the sensitivity of the 5-CSRTT to detect pharmacologicallyinduced impulsive action.