Targeted lentiviral vectors pseudotyped with the Tupaia paramyxovirus glycoproteins.

Lentiviral vectors are vectors of choice for many gene therapy applications. Recently, efficient targeting of lentiviral vectors pseudotyped with the Measles virus (MV) glycoproteins has been reported. However, MV antibodies in patients might limit the clinical use of these vectors. We demonstrate here that lentiviral vectors can also be pseudotyped with the glycoproteins of Tupaia paramyxovirus (TPMV), the hemagglutinin (H) and fusion (F) protein. As this animal paramyxovirus has no known close relatives in humans, we do not expect TPMV antibodies in patients. Because TPMV normally does not infect human cells, 'detargeting' from natural receptors is unnecessary. Similar to the MV system, TPMV glycoproteins can mediate targeted cell entry by displaying different single-chain antibodies (scAb) directed against surface molecules on target cells on the viral hemagglutinin. We generated a panel of H and F proteins with truncated cytoplasmic tails and determined the variants that efficiently pseudotyped lentiviral vectors. The B-cell marker CD20 was used as a model antigen, and CD20-targeted TPMV vectors selectively transduced CD20-positive cells, including quiescent primary human B-cells. Lentiviral vectors pseudotyped with targeted TPMV envelope proteins might be a valuable vector choice when systemic application of targeted lentiviral vectors in humans is required.

A feline model of spontaneously occurring autoimmune limbic encephalitis.

Autoimmune encephalitis (AE) is an important cause of encephalitis in humans and occurs at a similar rate to infectious encephalitis. It is frequently associated with antibodies against the extracellular domain of neuronal proteins. Among human AE, that with antibodies against leucine-rich glioma-inactivated 1 (LGI1) is one of the most prevalent forms, and was recently described in cats with limbic encephalitis (LE). In this study, we describe a large cohort (n = 32) of cats with AE, tested positive for voltage gated potassium channel (VGKC)-antibodies, of which 26 (81%) harboured LGI1-antibodies. We delineate their clinical and paraclinical features as well as long-term outcomes up to 5 years. Similar to human cases, most cats with LGI1-antibodies had a history of focal seizures (83%), clustering in the majority (88%), with interictal behavioural changes (73%). Among feline AE patients, there was no seizure type or other clinical characteristic that could distinguish LGI1-antibody positive from negative cats, unlike the pathognomic faciobrachial dystonic seizures seen in humans. Although six cats were euthanased in the first year for epilepsy-associated reasons, those attaining at least 1-year survival had good seizure control and quality of life with appropriate veterinary care and medication. Acute-phase immunotherapy (prednisolone) was given to the most severely unwell cases and its effect is retrospectively evaluated in 10 cats. Our data show LGI1-antibodies are an important cause of feline encephalitis, sharing many features with human AE. Further research should examine optimal therapeutic management strategies and the cause of LE in seronegative cats, building on paradigms established in the counterpart human disease.

A novel video-endoscope-guided myringotomy technique in dogs: Investigation in the value of vertical access to the tympanic membrane from beneath the patient - a pilot study.

Myringotomy is a well-accepted method for diagnosing and treating otitis media in dogs having an intact tympanic membrane. In a recent study, the contamination rate of middle ear aspirates from the external ear canal via myringotomy was 67%. To evaluate the iatrogenic contamination rate of the middle ear aspirates by material from the ear canal, using a novel technique: Vertical access to the tympanic membrane from beneath the patient. Thirty-six ears from 20 canine Fresh cadavers with a CT scan negative for otitis externa and otitis media were video-otoscopically flushed with the ear upside. The instillation of the fluorescent dye into the ear canal and immediate retrieval were performed. With the patient positioned on a custom-made fenestrated table plate, a modified video-otoscopically guided myringotomy approaching the tympanum vertically from underneath, was performed. Contamination rates were assessed by the visual detection of fluorescent dye within the aspirated fluid, either by yellow staining solely, or a positive fluorescence test. Cytology and microbial cultivation were accomplished. Middle ear sample contamination by the material from the ear canal was identified in 2 of 36 (5.55%) ears. Neither a change in colour nor fluorescence was detected in 34 of 36 (94.44%) middle ear samples. Sixteen of 36 (44.44%) external ear canal samples and 4 of 36 (11.11%) middle ear aspirates had positive bacterial culture. This novel technique is a promising method for middle ear material collection in patients with concurrent otitis externa.

Myringotomy in dogs: Contamination rate from the external ear canal - a pilot study.

Myringotomy for sample collection from the middle ear cavity for cytology and bacterial culture is considered a routine method to diagnose otitis media in dogs. The objective of this study was to determine the rate of contamination of middle ear aspirates with material from the external ear canal obtained by video-otoscopic guided myringotomy. In canine cadavers (n = 17) free from otitis externa the external ear canals were flushed under video-otoscopic control and a fluorescent dye was instilled. After removal of residual fluid a myringotomy was performed. If air was aspirated, 1 mL of saline was instilled through the same myringotomy needle into the middle ear cavity and re-aspirated. Contamination from the external ear canal was demonstrated by positive fluorescence of the aspirate. Bacterial cultures and cytological examinations of the external ear canals and middle ear cavities were performed. Data from 28 ears under investigation were included. In 19 of 28 middle ear aspirates (67.9%), clear yellow fluorescent fluid was obtained, indicating a contamination from the external ear canal. Microorganisms were detected in 4 of 26 middle ear samples (15.4%) and in 15 of 26 external ear canals (57.7%). Sample collection by myringotomy in this study was associated with a high contamination rate, implying that the suitability of this method for detection of otitis media in patients with concurrent otitis externa is questionable. Furthermore, the potential for iatrogenic spread of pathogenic microorganisms into the middle ear cavity needs to be considered.

Thirty-two component finite element models of a horse and donkey digit.

REASONS FOR PERFORMING STUDY: The finite element (FE) method is the most powerful modelling technique available to explicate the biomechanics of the digit. It has already proved to be of high value in human podiatry. However, accurate models of the complex anatomy of the horse and donkey digit are currently lacking. OBJECTIVES: To develop FE models of the horse and donkey digit from computed tomography data, including all functionally relevant anatomy, and to perform simulations to replicate prestrain in the flexor tendons and quasistatic weightbearing. METHODS: Computed tomography data of the front right digits were obtained under general anaesthesia. The anatomy was rationalised into 32 functional components. The FE models were generated using a forward engineering technique. Linear or nonlinear material properties were applied according to published data. Prestraining of the flexor tendons was achieved by z-direction displacement, and loading by the application of 1 x body mass. RESULTS: The resultant FE models comprised over 10(6) elements. Z-direction displacement of the digital flexor tendons to compensate for general anaesthesia relaxation gave von Mises stress levels up to 134 MPa for the deep and 0.56 MPa for the superficial in the horse and 0.78 MPa and 0.27 MPa in the donkey, respectively. Weightbearing resulted in capsular deformation patterns consistent with in vivo observations, and maximum stress levels of 1.46 MPa for the horse and 0.89 MPa for the donkey. CONCLUSION: These high resolution FE models could give new insight into the biomechanics of the equid digit and provide new data regarding stress and strain levels within the tissues of the digit that are unobtainable by other means. POTENTIAL RELEVANCE: Application of the FE modelling technique could enable investigation of the biomechanics of orthopaedic problems and may provide a mechanistic basis for enhanced preventative and remedial management and treatment.

Comparison of computed tomographic images of normal cranial and upper cervical lymph nodes with corresponding E12 plastinated-embedded sections in the dog.

To document normal lymph nodes on computed tomographic images, 102 scans were reviewed of dogs that had computed tomography (CT) of the head and upper cervical region. If lymph nodes were identified, symmetry, density, homogeneity, and size, as well as the relation to the surrounding fat tissue were noted. CT images with distinct asymmetric, enlarged, and obliterated lymph nodes were excluded. To improve the detailed anatomical topography, corresponding E12 plastinated-embedded sections of a dog were used. Compared with muscle tissue, normal lymph nodes were slightly hypodense and had a homogeneous parenchyma. Mandibular and retropharyngeal lymphocentres could be seen consistently and CT proved to be a useful method both to detect their presence and to assess their morphology. It was not possible to identify the parotid lymph node consistently because of the lack of tissue contrast at the lymph node-gland interface.

MRI and CT diagnosis of acute desmopathy of the lateral collateral sesmoidean (navicular) ligament and long-term outcome in a horse.

Clinical, magnetic resonance imaging (MRI) and computed tomography (CT) findings of acute desmopathy of the lateral collateral sesmoidean (navicular) ligament (CSL) in a 13-year-old Hanoverian mare are presented. On admission to the clinic the horse showed a grade 5/6 left front-limb lameness at the walk, pain on coffin joint manipulation, and coffin joint effusion. Despite a positive palmar digital nerve block, radiographs and ultrasonography did not indicate reasons for the severe clinical signs. However, MRI revealed damage to the CSL and bone marrow oedema of the navicular bone (NB), whereas a focal bone defect of the NB at the CSL insertion zone was demonstrated best by CT. The horse was managed with complete box rest and a fibreglass cast for four weeks followed by a controlled exercise program. Follow-up examination revealed no lameness at the trot three months later and the patient had fully recovered within six months.

Expression of G alpha 16, a G-protein alpha subunit specific for hematopoiesis in acute leukemia.

G-proteins are essential in signal transduction pathways. A G-protein alpha subunit termed G alpha 16 was found to be exclusively expressed in hematopoietic cell lines. In cells derived from patients, G alpha 16 expression has been detected in progenitor- and pre-B ALL cells and also in peripheral blood stem cells (PBSC). In this study, we analyzed G alpha 16 expression using a RT-PCR technique by testing elutriated blood cells from normal donors, PBSC from breast cancer patients and bone marrow or peripheral blood cells from acute leukemia patients. Both of two ALL patients and 15/16 AML patients expressed G alpha 16. In elutriation experiments, G alpha 16 expression was found in fractions containing the highest number of precursor cells but was absent in mature T and B cell fractions. In addition, CD34-enriched PBSC were positive for G alpha 16 expression. Further in vitro experiments using the cell line KG1 showed that G alpha 16 expression was not affected by the growth inhibiting hemoregulatory peptide pEEDCK which has a sequence homology present within G alpha 16. Taken together, these data demonstrate that G alpha 16 is expressed in various normal and malignant hematopietic progenitors but not in their differentiated counterparts. G alpha 16 could play a vital role in signal transduction pathways controlling proliferation in early normal and malignant hematopoiesis.

Severe hypercalcemia in a dog with a retained fetus and endometritis.

A 10-month-old sexually intact female German Shorthaired Pointer examined because of lethargy, episodes of fever, inappetence, and vomiting was found to have severe hypercalcemia. Results of laboratory testing, radiography, and ultrasonography excluded previously recognized causes of hypercalcemia in dogs. Instead, the dog was found to have purulent endometritis and an incompletely resorbed fetus. Treatment with fluids i.v., diuretics, and calcitonin failed to adequately reduce serum calcium concentration, but serum calcium concentration was normal within 4 days after the dog underwent an ovariohysterectomy. Retention of one or more fetuses and endometritis should be included in the differential diagnosis for dogs with hypercalcemia.

[Radiologically detectable changes in the radial annular ligament and the supinator muscle of the domestic cat (Felis catus)]. / Uber röntgenologisch nachweisbare Veränderungen in Ligamentum anulare radii und Musculus supinator bei der Hauskatze (Felis catus).

Elbow joints of a random sample of 59 cats were investigated by means of X-rays and dissection as well as histologically and by making cuts through synthetic resin-embedded joints. Bone-dense shadow craniolateral to the elbow joint was observed in X-ray images of 23 limbs (about 20%). Furthermore 8 of these 23 elbow joints (about 7%) showed signs of arthrotic alteration. Histologic examination revealed one or more centers of metaplasia of varying status of development of the annular ligament of the radius. Alterations varied from chondroid metaplasia to mineralisation and ossification. They turned out to be located in the area where the annular ligament of the radius crosses the tendon of origin of the supinator muscle and are considered to be a reaction of tough connective tissue to mechanical stress. Compressive forces seem to be less responsible than tensile stress. As soon as bone-dense shadow is observed in X-ray images, alterations have grown so far as the annular ligament of the radius as well as the tendon of origin of the supinator muscle are affected.

Validation study of an interpolation method for calculating whole lung volumes and masses from reduced numbers of CT-images in ponies.

Quantitative computer tomographic analysis (qCTA) is an accurate but time intensive method used to quantify volume, mass and aeration of the lungs. The aim of this study was to validate a time efficient interpolation technique for application of qCTA in ponies. Forty-one thoracic computer tomographic (CT) scans obtained from eight anaesthetised ponies positioned in dorsal recumbency were included. Total lung volume and mass and their distribution into four compartments (non-aerated, poorly aerated, normally aerated and hyperaerated; defined based on the attenuation in Hounsfield Units) were determined for the entire lung from all 5 mm thick CT-images, 59 (55-66) per animal. An interpolation technique validated for use in humans was then applied to calculate qCTA results for lung volumes and masses from only 10, 12, and 14 selected CT-images per scan. The time required for both procedures was recorded. Results were compared statistically using the Bland-Altman approach. The bias ± 2 SD for total lung volume calculated from interpolation of 10, 12, and 14 CT-images was -1.2 ± 5.8%, 0.1 ± 3.5%, and 0.0 ± 2.5%, respectively. The corresponding results for total lung mass were -1.1 ± 5.9%, 0.0 ± 3.5%, and 0.0 ± 3.0%. The average time for analysis of one thoracic CT-scan using the interpolation method was 1.5-2 h compared to 8 h for analysis of all images of one complete thoracic CT-scan. The calculation of pulmonary qCTA data by interpolation from 12 CT-images was applicable for equine lung CT-scans and reduced the time required for analysis by 75%.

[Comparison of susceptibility artifacts generated by microchips with different geometry at 1.5 Tesla magnet resonance imaging. A phantom pilot study referring to the ASTM standard test method F2119-07]. / Größenvergleich von Suszeptibilitätsartefakten verursacht durch Mikrochips unterschiedlicher Geometrie am 1,5-Tesla-Magnetresonanztomographen. Phantompilotstudie unter Berücksichtigung der ASTM-Standardtestmethode F2119-07.

OBJECTIVE: Ferromagnetic material in microchips, used for animal identification, causes local signal increase, signal void or distortion (susceptibility artifact) on MR images. To measure the impact of microchip geometry on the artifact's size, an MRI phantom study was performed. MATERIAL AND METHODS: Microchips of the labels Datamars®, Euro-I.D.® and Planet-ID® (n = 15) were placed consecutively in a phantom and examined with respect to the ASTM Standard Test Method F2119-07 using spin echo (TR 500 ms, TE 20 ms), gradient echo (TR 300 ms, TE 15 ms, flip angel 30°) and otherwise constant imaging parameters (slice thickness 3 mm, field of view 250 x 250 mm, acquisition matrix 256 x 256 pixel, bandwidth 32 kHz) at 1.5 Tesla. Image acquisition was undertaken with a microchip positioned in the x- and z-direction and in each case with a phase-encoding direction in the y- and z-direction. The artifact size was determined with a) a measurement according to the test method F2119-07 using a homogeneous point operation, b) signal intensity measurement according to Matsuura et al. and c) pixel counts in the artifact according to Port and Pomper. RESULTS: There was a significant difference in artifact size between the three microchips tested (Wilcoxon p = 0.032). A two- to three-fold increase in microchip volume generated an up to 76% larger artifact, depending on the sequence type, phase-encoding direction and chip position to B0. CONCLUSION AND CLINICAL RELEVANCE: The smaller the microchip geometry, the less is the susceptibility artifact. Spin echoes (SE) generated smaller artifacts than gradient echoes (GE). In relation to the spatial measurement of the artifact, the switch in phase-encoding direction had less influence on the artifact size in GE- than in SE-sequences. However, the artifact shape and direction of SE-sequences can be changed by altering the phase. The artifact size, caused by the microchip, plays a major clinical role in the evaluation of MRI from the head, shoulder and neck regions.

Foraminal and paraspinal extraforaminal attachments of the sixth and seventh lumbar spinal nerves in large breed dogs.

Fresh cadaveric lumbar spines of 20 adult large breed dogs were used to study the sixth and seventh lumbar spinal nerves along their course through their respective intervertebral foramen. The relationship between the periosteum lining the vertebral canal (endorhachis; peridural membrane) and the vessels inside the vertebral canal, and the relationship between the nerves and the wall of the intervertebral foramen and the extraspinal suspensory apparatus were investigated. Each intervertebral foramen contained a fibrous septum that divided it into two sub-compartments by connecting the fibrous capsule of the facet joints with the intervertebral disc and the adjoining vertebral body. The lumbar nerves and the main artery passed through the cranial sub-compartment and the main vein passed through the caudal sub-compartment. In all cases, there was a circumneural sleeve that connected the ventral branches of the lumbar nerves extraspinally with the fibrous capsule of the facet joints dorsally, the fibrous septum caudally, and the caudal vertebral notch and accessory process cranioventrally. The deep layer of the circumneural sleeve was formed by the periosteum lining the vertebral canal pouching laterally through the intervertebral foramen; the superficial (lateral) layer was formed by the deep sheet of the thoracolumbar fascia. The deep sheet of the thoracolumbar fascia continued cranially and caudally to the circumneural sleeve to attach it to the vertebral body and the intervertebral disc. Regional and individual differences were noted in the composition and length of the circumneural sleeve. The potential biomechanical and clinical roles of these variations are discussed.

MR anatomy of salivary glands in the dog.

This retrospective analysis documented the magnetic resonance imaging (MRI) appearance of normal salivary glands based on 101 studies in dogs with no detectable disease in the splanchnocranium. Surface, signal intensity, homogeneity, structure, symmetry and the relationship of glands to surrounding tissues were noted, and gland topography was assessed with E12 plastinated embedded sections. Signal intensity of salivary glands was isointense (7-40%) to hyperintense (60-90%) to muscle tissue on T1- and hyperintense on T2-weighted images. Salivary glands had an increased T1 signal after contrast medium was applied. Salivary gland structure appeared homogeneous in mandibular and major sublingual glands and heterogeneous in zygomatic and parotid glands. Consistent landmarks were the external auditory canal for parotid glands, the digastric muscle for mandibular and major sublingual glands, and the pterygopalatine fossa for zygomatic glands. The minor sublingual and ventral buccal glands could not be localized with low-field MRI.

CT sialography in the dog - a cadaver study.

To document computed tomographic topography of salivary glands and their ducts in dogs, a retrograde filling with methylcellulose and iodinated contrast medium was performed in three cadavers. Demarcation of the parotid, mandibular and zygomatic glands was achieved. Surrounding structures were imaged without beam hardening artefacts. Landmarks for the parotid, mandibular and zygomatic glands were the external acoustic canal, the mandibular angle and the pterygopalatine fossa, respectively. Sialograms of the parotid, mandibular and zygomatic ducts were achieved, whereas neither the sublingual glands nor their ducts could be contrasted. The images provide a three-dimensional visualization of the salivary glands and their ducts.

Spongy degeneration with cerebellar ataxia in Malinois puppies: a hereditary autosomal recessive disorder?

BACKGROUND: There is a high incidence of hereditary degenerative diseases of the central nervous system in purebred dogs. HYPOTHESIS: Cerebellar ataxia in Malinois puppies, caused by degenerative changes that predominate in cerebellar nuclei and the granular cell layer, is a hereditary disorder that is distinct from cerebellar cortical abiotrophies. ANIMALS: Thirteen Malinois puppies with cerebellar ataxia. METHODS: Retrospective study. Records of Malinois puppies with spongy degeneration of the cerebellar nuclei were analyzed including clinical signs, histopathological changes, and pedigree data. RESULTS: Signs of cerebellar dysfunction were observed in puppies of both sexes from 5 different litters (1995-2009) of phenotypically normal parents. Clinical signs started before the age of 2 months and resulted in euthanasia of all puppies by the age of 13 weeks. Histopathology disclosed marked bilateral spongy degeneration of the cerebellar nuclei and vacuoles in the granular cell layer and foliate white matter of the cerebellum. In some puppies, discrete vacuoles in gray and white matter were present in other parts of the brain. Furthermore, spheroids and dilated myelin sheaths were observed. Pedigree data and segregation frequency support an autosomal recessive hereditary disorder. CONCLUSIONS AND CLINICAL IMPORTANCE: Malinois suffer from a hereditary spongiform degeneration that predominates in the cerebellum and causes an early onset of clinical signs with unfavorable prognosis. Future efforts should increase awareness among veterinarians and breeders and aim to identify underlying metabolic mechanisms and the affected genes.

Opacity and lucency sign of the seventh lumbar vertebra in dogs.

A curvilinear opacity of the seventh vertebral arch and an adjacent focal lucency of the seventh lumbar vertebral body were incidentally noted in routine radiographs. The aim of this study was to analyze this radiographic absorption pattern using a sample of 51 dogs. Images of the bony surface of three macerated lumbosacral junctions were compared to their laterolateral radiographs and computed tomographic (CT) scans. Additional 48 lateral radiographic projections were reviewed for presence or absence of L7 opacity and lucency, length of the L6 and L7 vertebral bodies, vertebral disc disease, osteochondrosis, or spondylosis. Retrieved data were compiled and statistically analyzed. Radiographs and CT scans of the macerated bones revealed that the L7 pedicle had thickened layers of compact bone and the adjacent vertebral notch varying depth and extension. Superimposition of the pedicle on the vertebral body resulted in a curvilinear opacity (L7 opacity), the vertebral notch caused the focal lucency (L7 lucency). These findings were present in 69% of the reviewed radiographs and were found to be significantly associated with L6-L7 length ratios of less than 0.86 (P = 0.003). It is hypothesized that the changes correspond with normal anatomic morphology of the seventh lumbar vertebra plus adaptive bone remodelling. The importance of this study is that it could help avoid misinterpretation of this appearance as a pathologic condition.

Cervical spinal chordoma with chondromatous component in a dog.

A 7-year-old male Belgian Shepherd dog was presented with sudden onset of lateral recumbency and tetraparesis. At the level of the third cervical vertebra, magnetic resonance imaging demonstrated an intrameningeal and intramedullary mass lesion. The animal was subsequently euthanatized. A necropsy revealed a semitranslucent solid mass infiltrating dorsal and ventral dura mater and the spinal cord. Histologic examination revealed a lobulated pleomorphic mass, mainly resembling undifferentiated cartilage interspersed by spindle-shaped and polygonal cells with highly vacuolated cytoplasm (physaliphorous cells). Immunohistochemistry of the tumor cells demonstrated dual expression of vimentin and cytokeratin. Based on the histologic and immunohistochemical results, the diagnosis of a chordoma with chondromatous component was made.

Computed tomographic anatomy of the canine temporal bone.

This study aims to identify and describe structures of the temporal bone in normal dogs that can be depicted using computed tomography (CT). Five normal dogs of different breeds and both sexes ranging from 0.5 to 8 years were used as the study material. CT images of 2 mm thickness without abnormalities in the temporal bone were reviewed and analysed in bone and soft tissue settings. All images were compared with corresponding E 12 plastic-embedded cross sections of 3 mm thickness, which were acquired from anatomic cross sections of frozen beagles. The sections were provided from the Department of Pathobiology (Institute of Anatomy). CT is able to image all relevant bony structures within the temporal bone. Concerning soft tissue structures, CT allows their localization by using bony markers, i.e. the internal acustic meatus for the cranial nerves VII and VIII or the canals for the internal carotid artery and the temporal sinus. The provided reference images are meant to improve orientation within the complex anatomy of the temporal bone. On condition that slice thickness is equal to or smaller than 2 mm and bony setting is supplied, CT is able to visualize all relevant structures within the temporal bone. However, in clinical settings the combined look on air, liquids and bone as well as the contrast enhancement of soft tissues might prefer magnetic resonance imaging to CT.