p19Arf sensitizes B16 melanoma cells to interferon-ß delivered via mesenchymal stem cells in vitro.

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-ß (IFNß) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNß was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNß production. MSCs used to produce IFNß inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNß produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNß and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

p19Arf sensitizes B16 melanoma cells to interferon-β delivered via mesenchymal stem cells in vitro

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-β (IFNβ) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNβ was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNβ production. MSCs used to produce IFNβ inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNβ produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNβ and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

Temperature dependence of the Raman spectrum of 1-(4-chlorophenyl)-3-(2-thienyl)prop-2-en-1-one.

The heterocyclic chalcone containing thiophene ring 1-(4-chlorophenyl)-3-(2-thienyl)prop-2-en-1-one, C13H9ClOS was synthesized and investigated using experimental techniques such as nuclear magnetic resonance (1H and 13C NMR), Fourier transform infrared spectroscopy (FTIR) at room temperature, differential scanning calorimeter (DSC) from room temperature to 500K and Raman scattering at the temperature range 10-413K in order to study its structure and vibrational properties as well as stability and possible phase transition. Density functional theory (DFT) calculations were performed to determine the vibrational spectrum viewing to improve the knowledge of the material properties. A reasonable agreement was observed between theoretical and experimental Raman spectrum taken at 10K since anharmonic effects of the molecular motion is reduced at low temperatures, leading to a more comprehensive assignment of the vibrational modes. Increasing the temperature up to 393K, was observed the typical phonon anharmonicity behavior associated to changes in the Raman line intensities, line-widths and red-shift, in special in the external mode region, whereas the internal modes region remains almost unchanged due its strong chemical bonds. Furthermore, C13H9ClOS goes to melting phase transition in the temperature range 393-403K and then sublimates in the temperature range 403-413K. This is denounced by the disappearance of the external modes and the absence of internal modes in the Raman spectra, in accordance with DSC curve. The enthalpy (ΔH) obtained from the integration of the endothermic peak in DSC curve centered at 397K is founded to be 121.5J/g.

Kinematic Magnetic Resonance Imaging for Evaluation of Disc-Associated Cervical Spondylomyelopathy in Doberman Pinschers.

BACKGROUND: The dynamic component of disc-associated cervical spondylomyelopathy (DA-CSM) currently is evaluated using traction magnetic resonance imaging (MRI), which does not assess changes in flexion and extension of the cervical vertebral column. In humans with cervical spondylotic myelopathy, kinematic MRI is used to identify dynamic compressions. HYPOTHESIS/OBJECTIVES: To evaluate the feasibility and utility of kMRI in Doberman Pinschers with DA-CSM using a novel positioning device. We hypothesized that kMRI would identify compressive lesions not observed with neutral positioning and change the dimensions of the spinal cord and cervical vertebral canal. ANIMALS: Nine client-owned Doberman Pinschers with DA-CSM. METHODS: Prospective study. After standard MR imaging of the cervical spine confirmed DA-CSM, dogs were placed on a positioning device to allow imaging in flexion and extension. Morphologic and morphometric assessments were compared between neutral, flexion, and extension images. RESULTS: Flexion was associated with improvement or resolution of spinal cord compression in 4/9 patients, whereas extension caused worsening of compressions in 6/9 patients. Extension identified 6 new compressive lesions and was significantly associated with dorsal and ventral compression at C5-C6 (P = .021) and C6-C7 (P = .031). A significant decrease in spinal cord height occurred at C6-C7 from neutral to extension (P = .003) and in vertebral canal height at C5-C6 and C6-C7 from neutral to extension (P = .011 and .017, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Our results suggest that kMRI is feasible and provides additional information beyond what is observed with neutral imaging, primarily when using extension views, in dogs with DA-CSM.

von Frey anesthesiometry to assess sensory impairment after acute spinal cord injury caused by thoracolumbar intervertebral disc extrusion in dogs.

Sensory threshold (ST) was measured using an electric von Frey anesthesiometer (VFA) in all limbs of 20 normal dogs and 29 dogs with acute thoracolumbar spinal cord injury (SCI) caused by spontaneous intervertebral disc extrusion. ST values were measured at three separate time points in normal dogs and on days 3, 10 and 30 following decompressive surgery in dogs with SCI. ST values were compared between groups and correlated with locomotor recovery in SCI-affected dogs. ST values were significantly higher (consistent with hypoalgesia) in the pelvic limbs of SCI-affected dogs at day 3, day 10 and day 30 when compared to normal dogs (P <0.05), while no significant difference in thoracic limb ST values was observed between groups. A progressive decrease in pelvic limb ST values occurred in SCI-affected dogs over time, consistent with improvement toward normal sensation or development of allodynia. This finding correlated inversely with locomotor score at 3 and 10 days after surgery. A significant decline in ST values across testing sessions was observed for all limbs of normal and SCI-affected dogs and may be related to patient acclimation, operator training effect, or effect of analgesic medications. This study supports the feasibility of VFA to assess differences in ST between normal and SCI-affected dogs. However, future studies must focus on techniques to minimize or compensate for clinical, environmental and behavioral factors which may impact ST values in the clinical setting.

A simplified method of walking track analysis to assess short-term locomotor recovery after acute spinal cord injury caused by thoracolumbar intervertebral disc extrusion in dogs.

The purpose of this study was to evaluate a simplified method of walking track analysis to assess treatment outcome in canine spinal cord injury. Measurements of stride length (SL) and base of support (BS) were made using a 'finger painting' technique for footprint analysis in all limbs of 20 normal dogs and 27 dogs with 28 episodes of acute thoracolumbar spinal cord injury (SCI) caused by spontaneous intervertebral disc extrusion. Measurements were determined at three separate time points in normal dogs and on days 3, 10 and 30 following decompressive surgery in dogs with SCI. Values for SL, BS and coefficient of variance (COV) for each parameter were compared between groups at each time point. Mean SL was significantly shorter in all four limbs of SCI-affected dogs at days 3, 10, and 30 compared to normal dogs. SL gradually increased toward normal in the 30 days following surgery. As measured by this technique, the COV-SL was significantly higher in SCI-affected dogs than normal dogs in both thoracic limbs (TL) and pelvic limbs (PL) only at day 3 after surgery. BS-TL was significantly wider in SCI-affected dogs at days 3, 10 and 30 following surgery compared to normal dogs. These findings support the use of footprint parameters to compare locomotor differences between normal and SCI-affected dogs, and to assess recovery from SCI. Additionally, our results underscore important changes in TL locomotion in thoracolumbar SCI-affected dogs.

Body conformation in Great Danes with and without clinical signs of cervical spondylomyelopathy.

It has been suggested that a combination of large head and long neck cause abnormal forces on the cervical vertebral column and are involved in the pathogenesis of cervical spondylomyelopathy (CSM) in Great Danes. The aim of this study was to compare the body conformation of 15 clinically normal and 15 CSM-affected Great Danes. There were no statistically significant differences between clinically normal and CSM-affected Great Danes in any body measurements. There were no significant associations between body conformation and the severity of neurological signs or cervical vertebral body dimensions determined by magnetic resonance imaging in CSM-affected Great Danes. The results of this study do not support the hypothesis that differences in body conformation related to head size, neck length, and body height and length, play a role in the pathogenesis of CSM in Great Danes.

Cervical vertebral trabecular bone mineral density in Great Danes with and without osseous-associated cervical spondylomyelopathy.

BACKGROUND: Great Danes (GDs) with osseous-associated cervical spondylomyelopathy (CSM) have osteoarthritis (OA) of the cervical vertebrae. OA is often associated with increases in bone mineral density (BMD) in people and dogs. HYPOTHESIS/OBJECTIVES: To compare the trabecular BMD of the cervical vertebrae between clinically normal (control) GDs and GDs with osseous-associated CSM by using computed tomography (CT). We hypothesized that the vertebral trabecular BMD of CSM-affected GDs would be higher than that of control GDs. ANIMALS: Client-owned GDs: 12 controls, 10 CSM affected. METHODS: Prospective study. CT of the cervical vertebral column was obtained alongside a calibration phantom. By placing a circular region of interest at the articular process joints, vertebral body, pedicles, and within each rod of the calibration phantom, trabecular BMD was measured in Hounsfield units, which were converted to diphosphate equivalent densities. Trabecular BMD measurements were compared between CSM-affected and control dogs, and between males and females within the control group. RESULTS: Differences between CSM-affected and control dogs were not significant for the articular processes (mean = -39; P = .37; 95% CI: -102 to 24), vertebral bodies (mean = -62; P = .08; 95% CI: -129 to 6), or pedicles (mean = -36; P = .51; 95% CI: -105 to 33). Differences between female and male were not significant. CONCLUSIONS AND CLINICAL IMPORTANCE: This study revealed no difference in BMD between control and CSM-affected GDs. Based on our findings no association was detected between cervical OA and BMD in GDs with CSM.

Cytokine concentrations in the cerebrospinal fluid of great danes with cervical spondylomyelopathy.

BACKGROUND: Chronic inflammation is involved in the pathogenesis of human cervical spondylotic myelopathy and could also play a role in cervical spondylomyelopathy (CSM) in dogs. HYPOTHESIS/OBJECTIVES: That cerebrospinal fluid (CSF) cytokine concentrations would differ between clinically normal (control) and CSM-affected Great Danes (GDs), with affected GDs showing higher levels of inflammatory cytokines, such as interleukin (IL)-6 and monocyte chemoattractant protein-1/chemokine ligand 2 (MCP-1/CCL2). ANIMALS: Client-owned GDs: 15 control, 15 CSM-affected. METHODS: Prospective study. Dogs underwent cervical vertebral column magnetic resonance imaging and collection of CSF from the cerebellomedullary cistern. Cytokine concentrations were measured using a commercially available canine multiplex immunoassay. Cytokine concentrations were compared between groups. Associations with the administration of anti-inflammatory medications, disease duration and severity, severity of spinal cord (SC) compression, and SC signal changes were investigated in affected GDs. RESULTS: Affected GDs had significantly lower MCP-1/CCL2 (mean 138.03 pg/mL, 95% confidence interval [CI] = 114.85-161.20) than control GDs (212.89 pg/mL, 95% CI = 165.68-260.11, P = .028). In affected GDs, MCP-1/CCL2 concentrations correlated inversely with the severity of SC compression. There were no associations with administration of anti-inflammatory medications, disease duration, or disease severity. IL-6 concentrations were significantly higher (2.20 pg/mL, 95% CI = 1.92-2.47, P < .001) in GDs with SC signal changes. CONCLUSIONS AND CLINICAL IMPORTANCE: Lower MCP-1/CCL2 in CSM-affected GDs might compromise clearance of axonal and myelin debris, delay axon regeneration, and affect recovery. Higher IL-6 in CSM-affected GDs with SC signal changes suggests more severe inflammation in this group.

Cervical spondylomyelopathy in Great Danes: a magnetic resonance imaging morphometric study.

Morphometric investigations comparing normal and affected animals increase our understanding of spinal diseases in dogs. The aim of this study was to generate morphometric data for osseous-associated cervical spondylomyelopathy (CSM) in Great Danes (GDs). Magnetic resonance imaging (MRI) morphometric features of the cervical vertebral column of GDs with and without clinical signs of CSM were characterized and compared. Thirty client-owned GDs were prospectively enrolled, including 15 clinically normal and 15 CSM-affected GDs. All dogs underwent MRI of the cervical to thoracic vertebral column (C2-C3 through T1-T2). Areas of the cranial and caudal articular processes, and the height, width and areas of the vertebral canal and spinal cord were determined. Middle foraminal heights were measured. Intervertebral disc width was measured before and after traction. Intraobserver and interobserver agreement were calculated. CSM-affected GDs had larger areas of the caudal articular processes from C2-C3 through T1-T2. In CSM-affected GDs, the vertebral canal and spinal cord areas were significantly smaller at C5-C6 and C6-C7, the vertebral canal width was significantly narrower at C6-C7 and C7-T1, and the spinal cord width was significantly narrower at C5-C6 and C6-C7. Middle foraminal height was smaller in CSM-affected GDs from C3-C4 through C7-T1. Neutral intervertebral disc widths were smaller in CSM-affected GDs. It was concluded that the cervical vertebral canal dimensions are significantly different between normal and CSM-affected GDs. Absolute vertebral canal stenosis and severe foraminal stenosis involving the cervical vertebrae distinguish CSM-affected from clinically normal GDs. These findings are relevant to the pathogenesis of osseous-associated CSM and should be taken into consideration when performing imaging studies and planning surgery.

Transcranial magnetic motor evoked potentials in Great Danes with and without clinical signs of cervical spondylomyelopathy: association with neurological findings and magnetic resonance imaging.

Transcranial magnetic motor evoked potentials (TMMEPs) assess the functional integrity of the descending motor pathways, which are typically compromised in canine cervical spondylomyelopathy (CSM). The objective of this prospective study was to establish the reference ranges of TMMEP latency and amplitude in clinically normal (control) Great Danes (GDs), compare TMMEPs obtained in GDs with and without CSM, and determine whether there is any association between TMMEP data and severity of neurological signs or magnetic resonance imaging (MRI) findings. Twenty-nine client-owned GDs were enrolled (15 controls, 14 CSM-affected). All dogs underwent TMMEPs under sedation, and latencies and amplitudes were recorded from the extensor carpi radialis (ECR) and cranial tibial (CT) muscles. MRI of the cervical vertebral column was performed to evaluate the presence and severity of spinal cord (SC) compression, and the presence of SC signal changes. ECR and CT latencies were significantly longer in CSM-affected than control GDs. No significant differences between groups were found for amplitudes or neuronal path lengths. For the CT TMMEPs, CSM-affected GDs with moderate and severe clinical signs had significantly longer latencies than those with mild clinical signs. Significantly longer CT latencies were found in dogs with moderate and severe SC compression compared with dogs with mild compression. CT TMMEPs could not be recorded in 7/9 CSM-affected GDs with SC signal changes. These results provide a reference range for TMMEPs of clinically normal GDs. The use of TMMEPs is a valid ancillary test to assess the integrity of motor pathways in GDs with CSM.

Force plate gait analysis in Doberman Pinschers with and without cervical spondylomyelopathy.

BACKGROUND: The most accepted means of evaluating the response of a patient with cervical spondylomyelopathy (CSM) to treatment is subjective and based on the owner and clinician's perception of the gait. OBJECTIVE: To establish and compare kinetic parameters based on force plate gait analysis between normal and CSM-affected Dobermans. ANIMALS: Nineteen Doberman Pinschers: 10 clinically normal and 9 with CSM. METHODS: Force plate analysis was prospectively performed in all dogs. At least 4 runs of ipsilateral limbs were collected from each dog. Eight force platform parameters were evaluated, including peak vertical force (PVF) and peak vertical impulse (PVI), peak mediolateral force (PMLF) and peak mediolateral impulse, peak braking force and peak braking impulse, and peak propulsive force (PPF) and peak propulsive impulse. In addition, the coefficient of variation (CV) for each limb was calculated for each parameter. Data analysis was performed by a repeated measures approach. RESULTS: PMLF (P = .0062), PVI (P = .0225), and PPF (P = .0408) were found to be lower in CSM-affected dogs compared with normal dogs. Analysis by CV as the outcome indicated more variability in PVF in CSM-affected dogs (P = 0.0045). The largest difference in the CV of PVF was seen in the thoracic limbs of affected dogs when compared with the thoracic limbs of normal dogs (P = 0.0019). CONCLUSIONS AND CLINICAL IMPORTANCE: The CV of PVF in all 4 limbs, especially the thoracic limbs, distinguished clinically normal Dobermans from those with CSM. Other kinetic parameters less reliably distinguished CSM-affected from clinically normal Dobermans.

Three-dimensional kinematic gait analysis of Doberman Pinschers with and without cervical spondylomyelopathy.

BACKGROUND: The optimal treatment of cervical spondylomyelopathy (CSM) is controversial, with the owner's and clinician's perception of gait improvement often being used as outcome measures. These methods are subjective and suffer from observer bias. OBJECTIVES: To establish kinematic gait parameters utilizing digital motion capture in normal Doberman Pinschers and compare them with CSM-affected Dobermans. ANIMALS: Nineteen Doberman Pinschers; 10 clinically normal and 9 with CSM. METHODS: All dogs were enrolled prospectively and fitted with a Lycra(®) body suit, and motion capture was performed and used to reconstruct a 3-D stick diagram representation of each dog based on 32 reflective markers, from which several parameters were measured. These included stride duration, length, and height; maximal and minimal spinal angles; elbow and stifle flexion and extension; and maximum and minimum distances between the thoracic and pelvic limbs. A random-effects linear regression model was used to compare parameters between groups. RESULTS: Significant differences between groups included smaller minimum (mean = 116 mm; P = .024) and maximum (mean = 184 mm; P = .001) distance between the thoracic limbs in CSM-affected dogs. Additionally, thoracic limb stride duration was also smaller (P = .009) in CSM-affected dogs (mean = 0.7 seconds) when compared with normal dogs (mean = 0.8 seconds). In the pelvic limbs, the average stifle flexion (mean = 100°; P = .048) and extension (mean = 136°; P = .009), as well as number of strides (mean = 2.7 strides; P = .033) were different between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings suggest that computerized gait analysis reveals more consistent kinematic differences in the thoracic limbs, which can be used as future objective outcome measures.

Current insights and controversies in the pathogenesis and diagnosis of disc-associated cervical spondylomyelopathy in dogs.

Disc-associated cervical spondylomyelopathy (DA-CSM) is the most common cause of cervical spondylomyelopathy in dogs. In this condition, progressive caudal cervical spinal cord compression is typically caused by protrusion of one or more intervertebral discs. This disc-associated compression is sometimes seen in combination with mild vertebral abnormalities and dorsal compression resulting from ligamentum flavum hypertrophy. The intervertebral disc space between the sixth (C6) and seventh (C7) cervical vertebrae is most commonly affected. Although several large breed dogs can be affected, the adult to older dobermann is overrepresented. Clinical signs vary from cervical hyperaesthesia to tetraplegia. Dogs can present with a chronic progressive or an acute onset of clinical signs. Many aspects of this multifactorial neurological syndrome are not completely understood and are the subject of controversy and debate. Although several factors have been proposed, the underlying pathology and aetiology remain unknown. Recently, new insights have been gained in the pathogenesis, diagnosis and treatment of this challenging neurological syndrome. This review outlines current controversies and new developments concerning the pathogenesis and diagnosis of DA-CSM.

Micromorphometry and cellular characteristics of the canine cervical intervertebral discs.

BACKGROUND: Dogs have a high prevalence of disc-associated cervical spinal cord disorders. Despite this, there are no descriptions of the micromorphometry or cellular characteristics of canine cervical intervertebral discs. HYPOTHESIS/OBJECTIVES: To compare micromorphometric and cellular characteristics at 4 disc regions (outer annulus [OA], inner annulus [IA], transitional zone [TZ], nucleus pulposus [NP]). We hypothesized that measurements would differ between chondrodystrophic (CH) and nonchondrodystrophic (NCH) dogs. ANIMALS: Six CH dogs and 6 NCH dogs, under 3 years old. METHODS: Histologic sections of all cervical discs from each dog were examined. Micromorphometric variables included relative ratio of ventral to dorsal annulus fibrosus (AF), number and mean width of AF fibers, and relative percentage of NP. Cellular variables included cell density, morphology, and grouping. RESULTS: The NP from CH dogs was smaller, more rounded, and more dorsally located compared with that from NCH dogs. The NP occupied a greater proportion of the disc in NCH dogs compared with CH dogs (27.7 versus 15.8%; P<.001). The ratio of ventral to dorsal AF was approximately 3:1 in the CH group and 2:1 in the NCH group. Cellular variables were region dependent. Cell density was 2.4-fold higher in the OA compared with the NP. Approximately 50 of NP cells and 4.5% of OA cells occurred in groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Micromorphometric variables differed by CH status. Cellular variables differed by disc region. Our findings potentially can explain the high incidence of intervertebral disc extrusions in CH dogs compared with NCH dogs.

MRI measurement of the canine auditory pathways and relationship with brainstem auditory evoked responses.

The objective of this study was to determine direct measurements of auditory pathways by magnetic resonance imaging (MRI) during the growth period of healthy Beagles, and to discover how canine brainstem auditory evoked response (BAER) latencies vary in relation to these MRI measurements. Eighty healthy Beagles were tested at eight, 16 and 52 weeks of age (stages 1, 2, 3, respectively) with BAER and brain MRI. The BAER interpeak latency (IPL) II-V and brain MRI neural generators of BAER waves II and V were identified. A linear distance was calculated in millimeters in order to determine the approximate length of auditory pathways. Sensory nerve conduction velocity (SNCV) of the auditory pathway between peak II and peak V was calculated for each group. A significant difference was observed between brain MRI distances among the three stages. Mean BAER IPL II-V were not significantly different between the three stages. The progressive growth of the skull and brain witnessed by the progressive increased distance of the MRI auditory pathways between peak II and peak V was not associated with a progressive maturation of the BAER IPL II-V. The SNCV of the auditory pathway between peak II and peak V was 6.14 m/sec for group 1; 6.76 m/sec for group 2; and 7.32 m/sec for group 3.