Search

Some children with proven intrauterine Zika virus (ZIKV) infection who were born asymptomatic subsequently manifested neurodevelopmental delays, pointing to impairment of development perinatally and postnatally. To model this, we infected postnatal day (P) 5-6 (equivalent to the perinatal period in humans) susceptible mice with a mammalian cell-propagated ZIKV clinical isolate from the Brazilian outbreak in 2015. All infected mice appeared normal up to 4 days post-intraperitoneal inoculation (dpi), but rapidly developed severe clinical signs at 5-6 dpi. All nervous tissue examined at 5/6 dpi appeared grossly normal. However, anti-ZIKV positive cells were observed in the optic nerve, brain, and spinal cord; predominantly in white matter. Co-labeling with cell type specific markers demonstrated oligodendrocytes and astrocytes support productive infection. Rarely, ZIKV positive neurons were observed. In spinal cord white matter, which we examined in detail, apoptotic cells were evident; the density of oligodendrocytes was significantly reduced; and there was localized microglial reactivity including expression of the NLRP3 inflammasome. Together, our observations demonstrate that a clinically relevant ZIKV isolate can directly impact oligodendrocytes. As primary oligodendrocyte cell death can lead later to secondary autoimmune demyelination, our observations may help explain neurodevelopmental delays in infants appearing asymptomatic at birth and commend lifetime surveillance.

Canine degenerative myelopathy (DM) is a progressive neurological disorder that may be considered to be a large animal model for specific forms of the fatal human disease, familial amyotrophic lateral sclerosis (fALS). DM is associated with a c118GA mutation of the superoxide dismutase 1 (Sod1) gene, and a significant proportion of cases are inherited in an autosomal recessive manner in contrast to the largely, but not exclusively, dominant mode of inheritance in fALS. The consensus view is that these Sod1/SOD1 mutations result in a toxic gain of function but the mechanisms remain unclear. Here we used an in vitro neuroblastoma cell line transfection system to monitor wild-type and mutant forms of SOD1 fusion proteins containing either a Cherry or an enhanced green fluorescent protein (EGFP) tag. These fusion proteins retained SOD1 enzymatic activity on a native gel assay system. We demonstrate that SOD1 aggregate density is significantly higher in DM transfectants compared to wild-type. In addition, we show by co-immunoprecipitation and confocal microscopy, evidence for a potential interaction between wild-type and mutant forms of SOD1 in co-transfected cells. While in vitro studies have shown SOD1 heterodimer formation in fALS models, this is the first report for DM SOD1. Therefore, despite for the majority of cases there is a difference in the mode of inheritance between fALS and DM, a similar interaction between wild-type and mutant SOD1 forms can occur. Clarifying the role of SOD1 in DM may also be of benefit to understanding the role of SOD1 in fALS.

Background The impact of epilepsy and its treatment on the quality of life (QoL) is considered an important part of treatment supervision in human epilepsy. Objectives To develop a list of key questions evaluating QoL in dogs with idiopathic epilepsy (IE) and their carers. Animals One hundred fifty-nine dogs with IE. Methods Cross-sectional study. An online project questionnaire was developed containing 90 QoL-associated questions that were initially allocated to 14 themes representing specific areas associated with the treatment and care of an epileptic dog. Principal component analysis was applied with the aim of refining the questionnaire to the least number of questions representing useful themes without loss of descriptive value. Carers were recruited by paper mail, primary practices, and canine epilepsy websites. Data were acquired from January to November 2011. Results Principal component analysis removed 54 questions, leaving 7 themes with 36 questions with a minimum Cronbach's alpha value of 0.7 indicating a good internal consistency: “Seizure severity and frequency”, “Adverse effects of antiepileptic drug (AED)”, “Restrictions on the carer's life”, “Frustrations over caring for a dog with IE”, “Carer distaste of AED adverse effects”, “Carer anxiety around the seizure event”, “Perceptions on rectal diazepam use”. Conclusions and Clinical Importance Principal component analysis successfully reduced the number of questions without loss in descriptive value. The remaining questions correlate well with each other in capturing valuable details about aspects of QoL and represent valuable key questions (EpiQoL) in the assessment of QoL for the carers of dogs with IE.

Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p

The Public Health Service Policy on the Humane Care and Use of Laboratory Animals and sound ethical practices require institutions to provide safe working environments for personnel working with animals; this mandate is achieved in part by establishing an effective animal care Occupational Health and Safety Program (OHSP). Land-grant institutions often face unique organizational challenges in fulfilling this requirement. For example, responsibilities for providing health and safety programs often have historically been dispersed among many different divisions scattered around the campus. Here we describe how our institutional management personnel overcame organizational structure and cultural obstacles during the formation of a comprehensive campus-wide animal care OHSP. Steps toward establishing the animal care OHSP included assigning overall responsibility, identifying all stakeholders, creating a leadership group, and hiring a fulltime Animal Care OHSP Specialist. A web-based portal was developed, implemented, and refined over the past 7 y and reflected the unique organizational structures of the university and the needs of our research community. Through this web-based portal, hazards are identified, risks are assessed, and training is provided. The animal care OHSP now provides easy mandatory enrollment, supports timely feedback regarding hazards, and affords enrollees the opportunity to participate in voluntary medical surveillance. The future direction and development of the animal care OHSP will be based on the research trends of campus, identification of emerging health and safety hazards, and ongoing evaluation and refinement of the program.

A structural transition in rare earth pyrohafnates, Ln{sub 2}Hf{sub 2}O{sub 7} (Ln=Y, La, Pr, Nd, Tb, Dy, Yb and Lu), has been identified. Neutron diffraction showed that the structure transforms from well-ordered pyrochloric to fully fluoritic through the lanthanide series from La to Lu with a corresponding increase in the position parameter x of the 48f (Fd3{sup Macron }m) oxygen site from 0.330 to 0.375. As evidenced by the selected area electron diffraction, La{sub 2}Hf{sub 2}O{sub 7}, Pr{sub 2}Hf{sub 2}O{sub 7} and Nd{sub 2}Hf{sub 2}O{sub 7} exhibited a well-ordered pyrocholoric structure with the presence of intense superlattice spots, which became weak and diffuse (in Dy{sub 2}Hf{sub 2}O{sub 7} and Tb{sub 2}Hf{sub 2}O{sub 7}) before disappearing completely as the series progressed towards the Lu end. High resolution electron microscopic studies showed the breakdown of the pyrochlore ordering in the form of antiphase domains resulting in diffused smoke-like superlattice spots in the case of Dy{sub 2}Hf{sub 2}O{sub 7} and Tb{sub 2}Hf{sub 2}O{sub 7}. - Graphical abstract: Transmission electron microscopic studies showed the ordered pyrochlore to defect fluorite transition in rare-earth pyrohafnates to occur via the formation of anti-phase domains to start with. Highlights: Black-Right-Pointing-Pointer Pyrochlore to fluorite structural transition in rare earthmore » pyrohafnates. Black-Right-Pointing-Pointer La{sub 2}Hf{sub 2}O{sub 7}, Pr{sub 2}Hf{sub 2}O{sub 7} and Nd{sub 2}Hf{sub 2}O{sub 7} showed well ordered pyrochlore structure. Black-Right-Pointing-Pointer Short range ordering in Dy{sub 2}Hf{sub 2}O{sub 7} and Tb{sub 2}Hf{sub 2}O{sub 7}. Black-Right-Pointing-Pointer Break down of pyrochlore ordering due to antiphase boundaries. Black-Right-Pointing-Pointer Rest of the series showed fluoritic structure.« less

Diffusion tensor imaging (DTI) is a powerful technique for the noninvasive assessment of the central nervous system. To facilitate the application of this technique to in vivo studies, we characterised a mouse model of the leukodystrophy, Pelizaeus-Merzbacher disease (PMD), comparing high-resolution ex vivo DTI findings with quantitative histological analysis of selected areas of the brain. The mice used in this study (Plp1-transgenic) carry transgenic copies of the Plp1 gene and are models for PMD as a result of gene duplication. Plp1 transgenic mice display a mild ataxia and experience frequent seizures around the time at which they were imaged. Axial (λ(1) ) and radial (RD) diffusivities and fractional anisotropy (FA) data were analysed using an exploratory whole-brain voxel-based method, a voxel-based approach using tract-based spatial statistics (TBSS), and by application of conventional region of interest (ROI) analyses to selected white matter tracts. Raw t value maps and TBSS analyses indicated widespread changes throughout the brain of Plp1-transgenic mice compared with the wild-type. ROI analyses of the corpus callosum, anterior commissure and hippocampal fimbria showed that FA was reduced significantly, whereas λ(1) and RD were increased significantly, in Plp1-transgenic mice compared with the wild-type. The DTI data derived from ROI analyses were subsequently compared with histological measures taken in the same regions. These revealed an almost complete absence of myelin, preservation of axons, marked astrocytosis and increased or unchanged cell densities. These data contribute to our growing understanding of the basis of anisotropic water diffusion in the normal and diseased nervous system.

A myopathy in the Great Dane dog with characteristic pathological and molecular features is reported. Young adults present with progressive weakness and generalised muscle atrophy. To better define this condition, an investigation using histopathology, confocal microscopy, biochemistry and microarray analysis was undertaken. The skeletal muscles of affected dogs exhibited increased oxidative fibre phenotype and core fibre lesions characterised by the disruption of the sarcomeric architecture and the accumulation of mitochondrial organelles. Affected muscles displayed co-ordinated expression of genes consistent with a slow-oxidative phenotype, which was possibly a compensatory response to chronic muscle damage. There was disruption of Z-lines in affected muscles which, at the molecular level, manifested as transcriptional dysregulation of several Z-line associated genes, including alpha-actinin, myotilin, desmin, vimentin and telethonin. The pathology of this canine myopathy is distinct from that of human central core myopathies that are characterised by cores devoid of mitochondria and by the presence of myofibrillar breakdown products.

Acute phase proteins (APPs) have become an important tool in the diagnosis, management and prognosis of inflammatory diseases in humans and are developing a similar utility in domestic species. Steroid-responsive meningitis arteritis (SRMA) is a well-recognised inflammatory disease of the dog, the diagnosis of which remains unsatisfactory based on clinical criteria and non-specific laboratory investigations. In this prospective pilot study the authors examined the acute phase response throughout the course of SRMA in serum and cerebrospinal fluid (CSF) by evaluating three key stages in disease management: presentation, treatment response and putative relapse. Serum APPs were found to be of value in supporting the diagnosis of SRMA and monitoring its treatment. C-reactive protein (CRP), serum amyloid-A (SAA), alpha-1-acid glycoprotein (AGP) and haptoglobin (Hp) all exhibited an increase above our laboratory reference range in nine patients at initial presentation. During treatment APPs decreased significantly compared to presentation except Hp which increased (Wilcoxon-Signed-Rank-test: CRP, SAA and AGP P

Steroid responsive meningitis-arteritis (SRMA) is a common disease of the dog with no specific ante-mortem diagnostic test and management is complicated by the requirement for prolonged treatment and the potential for relapse. The value of immunosuppressive prednisolone has been established but an optimal regime is not described. The lack of diagnostic and prognostic markers makes management frustrating. This study prospectively evaluates a protocol for the use of a prednisolone monotherapy in the management of SRMA. The utility of conventional and novel protein markers was evaluated by examining their expression at various milestones in the management of SRMA and by comparing this to their expression in other canine inflammatory and non-inflammatory central nervous system (CNS) diseases. This prospective study recruited twenty dogs with a clinical diagnosis of SRMA presenting to the University of Glasgow Small Animal Hospital between May 2006 and May 2008. These patients were enrolled on a strict prednisolone regimen with serum and cerebrospinal fluid samples taken at key points in the management of this disease. The protocol was successful at achieving resolution of clinical signs with no relapse in the follow up period in all 20 dogs. Clinical remission was achieved in all cases within 2 weeks of starting the protocol. When relapse occurred during the treatment schedule (4/20), restarting the treatment schedule led to resolution of the disease. The fact that dogs did not relapse post-treatment in this study is supportive of the value of this protocol in managing SRMA. Overall, the findings detailed in this thesis support the use of an immunosuppressive prednisolone monotherapy in the treatment of canine SRMA. Acute phase proteins are significantly elevated at initial presentation with the remission of clinical signs, induced by immunosuppressive therapy, reducing but not eliminating this increase in serum concentrations. APPs are of particular value in the identification of putative relapse. In contrast, serum and cerebrospinal fluid IgA provides valuable information at diagnosis but a persistent increase throughout the disease limits their value in the monitoring of therapy. This immunoglobulin was found to be also be elevated in the serum and cerebrospinal fluid of other inflammatory and non-inflammatory canine CNS diseases. A robust prognostic marker for SRMA remains elusive

The application of dielectrophoretic field-flow fractionation (depFFF) to the isolation of circulating tumor cells (CTCs) from clinical blood specimens was studied using simulated cell mixtures of three different cultured tumor cell types with peripheral blood. The depFFF method can not only exploit intrinsic tumor cell properties so that labeling is unnecessary but can also deliver unmodified, viable tumor cells for culture and/or all types of molecular analysis. We investigated tumor cell recovery efficiency as a function of cell loading for a 25 mm wide x 300 mm long depFFF chamber. More than 90% of tumor cells were recovered for small samples but a larger chamber will be required if similarly high recovery efficiencies are to be realized for 10 mL blood specimens used CTC analysis in clinics. We show that the factor limiting isolation efficiency is cell-cell dielectric interactions and that isolation protocols should be completed within approximately 15 min in order to avoid changes in cell dielectric properties associated with ion leakage.

Purpose Endocrine agents, such as letrozole, are established in the treatment of hormone-dependent breast cancer. However, response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Thus there is a need to identify novel markers predicting for response and to understand molecular mechanisms of resistance. Patients and Methods Sequential tumor biopsies were taken before and after 10 to 14 days of neoadjuvant treatment with letrozole in patients with estrogen receptor–rich breast cancer. Expression profiles on high-density microarray chips were then related to clinical responses as assessed from tumor volume measurements after 3 months of treatment. Results Of 52 patients, 37 (71%) were classified as having a clinical response to letrozole and 15 being clinically resistant. Bioinformatic analysis identified 205 covariables (69 baseline expression, 45 day 14 expression, and 91 change in expression with treatment) which differentiated between clinical responders and nonresponders. Hierarchical clustering using the variables separated responders and nonresponders into two distinct groups. Ontological assessment indicated that discriminating genes were enriched toward cellular biosynthetic processes. In particular, functional gene assessment showed ribosomal protein probes to have higher baseline expression in tumors responsive to letrozole and increased expression with treatment in nonresponding cases. Conclusion To our knowledge, this is the first study to describe genetic covariables and molecular processes discriminating between tumors clinically responsive and resistant to an aromatase inhibitor. The understanding of such molecular phenotypes will be important in optimizing the clinical use of aromatase inhibitors, both in terms of identifying responsive breast cancers and developing new agents to target resistance pathways.

Objectives: To characterise the clinical presentation and neurological abnormalities in dogs affected by aortic thromboembolism. Methods: The medical records of 13 dogs diagnosed with aortic thromboembolism as the cause of the clinical signs, and where a complete neurological examination was performed, were reviewed retrospectively. Results: The onset was acute in only four dogs, chronic in five dogs (with all of these presenting as exercise intolerance) or chronic with acute deterioration in four dogs. Dogs with an acute onset of clinical signs were more severely affected exhibiting neurological deficits, while dogs with a chronic onset of disease predominantly presented with the exercise intolerance and minimal deficits. The locomotor deficits included exercise intolerance with pelvic limb weakness (five of 13), pelvic limb ataxia (one of 13), monoparesis (two of 13), paraparesis (two of 13), non-ambulatory paraparesis (two of 13) and paraplegia (one of 13). There was an apparent male predisposition and the cavalier King charles spaniel was overrepresented. Clinical Significance: The rate of onset of clinical signs appears to segregate dogs affected by aortic thromboembolism into two groups, with different clinical characteristics and outcomes. Dogs with an acute onset of the clinical signs tend to be more severely affected, while dogs with a chronic onset predominantly present with exercise intolerance. It is therefore important to consider aortic thromboembolism as a differential diagnosis in dogs with an acute onset of pelvic limb neurological deficits and in dogs with longer standing exercise intolerance.

Duplication of PLP1, an X-linked gene encoding the major myelin membrane protein of the human CNS, is the most frequent cause of Pelizaeus-Merzbacher disease (PMD). Transgenic mice with extra copies of the wild type Plp1 gene, a valid model of PMD, also develop a dysmyelinating phenotype dependant on gene dosage. In this study we have examined the effect of increasing Plp1 gene dosage on levels of PLP/DM20 and on other representative myelin proteins. In cultured oligodendrocytes and early myelinating oligodendrocytes in vivo, increased gene dosage leads to elevated levels of PLP/DM20 in the cell body. During myelination, small increases in Plp1 gene dosage (mice hemizygous for the transgene) elevate the level of PLP/DM20 in oligodendrocyte soma but cause only minimal and transient effects on the protein composition and structure of myelin suggesting that cells can regulate the incorporation of proteins into myelin. However, larger increases in dosage (mice homozygous for the transgene) are not well tolerated, leading to hypomyelination and alteration in the cellular distribution of PLP/DM20. A disproportionate amount of PLP/DM20 is retained in the cell soma, probably in autophagic vacuoles and lysosomes whereas the level in myelin is reduced. Increased Plp1 gene dosage affects other myelin proteins, particularly MBP, which is transitorily reduced in hemizygous mice but consistently and markedly lower in homozygotes in both myelin and naïve or early myelinating oligodendrocytes. Whether the reduced MBP is implicated in the pathogenesis of dysmyelination is yet to be established.