An individually adjusted endurance test reveals differences in physical fitness between young and old Beagles.

OBJECTIVE: Aim was to establish an individually adapted endurance test for dogs on a treadmill, which takes the individual's physical condition into account. To check the applicability of the test, two age groups of clinically healthy beagles were examined. METHODS: A total of 10 clinically healthy Beagles were enrolled and divided in a younger (1-3 years, n = 5) and older group (> 8 years, n = 5). The individual comfort gait speed of each dog was determined on a treadmill with integrated force plates. A maximal time of 20 minutes at trot was set for the endurance test. The test was terminated prematurely if the dog showed signs of fatigue (massive panting, unwillingness to move further). Blood samples were taken at general examination (G), prior to (B) and post exercise (P) for determination of lactate level (LL), oxygen and carbon dioxide partial pressure (pO2, pCO2), bicarbonate (HCO3 -), base excess (BE) and pH. On each occasion (G, B, P) heart rate (HR) and respiratory rate (RR) were recorded. Additionally, vertical ground reaction forces (Fz) were analysed. RESULTS: The older dogs (age: 10.4 ±â€…0.89 years) completed the test with less speed and duration compared to the younger dogs (age: 2.4 ±â€…0.89 years), which managed to complete the maximum time. Lactate levels in the older dogs were higher than in the younger dogs at all timepoints of examination. Contrary to the younger dogs, there was no significant increase in the heart rate of the older dogs. Ground reaction forces were not significantly different between the groups. CONCLUSION AND CLINICAL RELEVANCE: Whereas standardised endurance tests allow for the comparison of fitness levels between dogs, an individually adjusted endurance test aims at objectively determining the physical fitness of the single dog taking into account its individual performance. Such a test allows to examine the individual performance development over time and to evaluate medicinal therapies or dietary measures, e. g. in aging dogs. HR, RR, LL, blood gases (pCO2, pO2) and acid-base metabolism (HCO3 -, BE, pH) were found to be appropriate parameters for determining the physical capacity of the dogs during endurance tests as these parameters change under physical stress and are indicative for the onset of fatigue.

Clinical Phenotypes of DMD Exon 51 Skip Equivalent Deletions: A Systematic Review.

BACKGROUND: Eteplirsen, the first FDA-approved RNA-modifying therapy for DMD, is applicable to ∼13% of patients with DMD. Because multiple exonic deletions are amenable to exon 51 skipping, the isoforms resulting from the various exon 51-skipped transcripts may vary in stability, function, and phenotype. OBJECTIVE/METHODS: We conducted a detailed review of dystrophinopathy published literature and unpublished databases to compile phenotypic features of patients with exon 51 "skip-equivalent" deletions. RESULTS: Theoretically, 48 different in-frame transcripts may result from exon 51 skipping. We found sufficient clinical information on 135 patients carrying mutations that would result in production of 11 (23%) of these transcripts, suggesting the remainder have not been identified in vivo. The majority had mild phenotypes: BMD (n = 81) or isolated dilated cardiomyopathy (n = 3). Particularly interesting are the asymptomatic (n = 10) or isolated hyperCKemia (n = 20) patients with deletions of exons 45- 51, 48- 51, 49- 51 and 50- 51. Finally, 16 (12%) had more severe phenotypes described as intermediate (n = 2) or DMD (n = 14), and 6 reports had no definitive phenotype. CONCLUSIONS: This review shows that the majority of exon 51 "skip-equivalent" deletions result in milder (BMD) phenotypes and supports that exon 51 skipping therapy could provide clinical benefit, although we acknowledge that other factors, such as age at treatment initiation or ongoing standard of care, may influence the degree of benefit.

Effect of antioxidants, mitochondrial cofactors and omega-3 fatty acids on telomere length and kinematic joint mobility in young and old shepherd dogs - A randomized, blinded and placebo-controlled study.

In dogs, decreasing telomere length is a biomarker for cellular aging. On a systemic level, aging affects the locomotor system in particular, leading to restricted joint mobility. As aging is thought to be related to oxidative stress, it may be counteracted by a diet enriched with antioxidants, mitochondrial cofactors and omega-3 fatty acids. This randomized, blinded and placebo-controlled study examined the influence of an accordingly enriched diet compared to a control diet on 36 young and 38 old shepherd dogs. At the outset, after 3 and after 6 months, mean and minimum telomere lengths were measured. Furthermore, minimum and maximum joint angles and range of motion of the shoulder, elbow, carpal, hip, stifle and tarsal joints were measured by computer-assisted gait analysis. A positive influence of the enriched diet on old dogs could be verified for minimum telomere length and all three parameters of the shoulder joint on the side with the higher vertical ground reaction force after 6 months. In the other joints there were less significant differences; in some cases they indicated a contrary influence of the enriched diet on young dogs, probably due to its reduced protein content. The greater effect of the enriched diet on minimum than on mean telomere length may be due to the higher preference of telomerase for short telomeres. The greater effect on shoulder joint mobility is explained by the greater influence of musculature and connective tissue in this joint. For elderly dogs it is advisable to feed these nutritional supplements.

Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.

Clinical use of submaximal treadmill exercise testing and assessments of cardiac biomarkers NT-proBNP and cTnI in dogs with presymptomatic mitral regurgitation.

Exercise intolerance is the first symptom of heart disease. Yet an objective and standardised method in canine cardiology to assess exercise capacity in a clinical setting is lacking. In contrast, exercise testing is a powerful diagnostic tool in humans, providing valuable information on prognosis and impact of therapeutic intervention. To investigate whether an exercise test reveals differences between dogs with early stage mitral regurgitation (MR) and dogs without cardiac disease, 12 healthy beagles (healthy group, HG) and 12 dogs with presymptomatic MR (CHIEF B1 / B2, patient group, PG) underwent a six-stage submaximal exercise test (ET) on a motorised treadmill. They trotted in their individual comfort speed for three minutes per stage, first without incline, afterwards increasing it by 4% for every subsequent stage. Blood samples were taken at rest and during two 3-minute breaks in the course of the test. Further samples were taken after the completion of the exercise test and again after a 3-hour recovery period. Measured parameters included heart rate, lactate and the cardiac biomarkers N-terminal pro-B-Type natriuretic peptide and cardiac Troponin I. The test was performed again under the same conditions in the same dogs three weeks after the first trial to evaluate individual repeatability. Cardiac biomarkers increased significantly in both HG and PG in the course of the test. The increase was more pronounced in CHIEF B1 / B2 dogs than in the HG. N-terminal pro-B-Type natriuretic peptide increased from 435 ± 195 to 523 ± 239 pmol/L (HG) and from 690 to 815 pmol/L (PG). cTnI increased from 0.020 to 0.024 ng/mL (HG) and from 0.06 to 0.08 ng/ml (PG). The present study provides a method to assess exercise-induced changes in cardiac biomarkers under clinical conditions. The increase of NT-proBNP and cTnI is more pronounced in dogs with early-stage MR than in healthy dogs. Results indicate that measuring the parameters before and after exercise is adequate and taking blood samples between the different stages of the ET does not provide additional information. Also, stress echocardiography was inconclusive. It can be concluded that exercise testing, especially in combination with measuring cardiac biomarkers, could be a helpful diagnostic tool in canine cardiology.

Three-dimensional inverse dynamics of the forelimb of Beagles at a walk and trot.

OBJECTIVE To perform 3-D inverse dynamics analysis of the entire forelimb of healthy dogs during a walk and trot. ANIMALS 5 healthy adult Beagles. PROCEDURES The left forelimb of each dog was instrumented with 19 anatomic markers. X-ray fluoroscopy was used to optimize marker positions and perform scientific rotoscoping for 1 dog. Inverse dynamics were computed for each dog during a walk and trot on the basis of data obtained from an infrared motion-capture system and instrumented quad-band treadmill. Morphometric data were obtained from a virtual reconstruction of the left forelimb generated from a CT scan of the same dog that underwent scientific rotoscoping. RESULTS Segmental angles, torque, and power patterns were described for the scapula, humerus, ulna, and carpus segments in body frame. For the scapula and humerus, the kinematics and dynamics determined from fluoroscopy-based data varied substantially from those determined from the marker-based data. The dominant action of scapular rotation for forelimb kinematics was confirmed. Directional changes in the torque and power patterns for each segment were fairly consistent between the 2 gaits, but the amplitude of those changes was often greater at a trot than at a walk. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that control of the forelimb joints of dogs is similar for both a walk and trot. Rotation of the forelimb around its longitudinal axis and motion of the scapula should be reconsidered in the evaluation of musculoskeletal diseases, especially before and after treatment or rehabilitation.

Comparative investigation of bone mineral density using CT and DEXA in a canine femoral model.

Bone density measurements using computed tomography (CT) instead of dual-energy X-ray absorptiometry (DEXA) are currently of great interest in human and veterinary medical research as it would be beneficial to use CT scans obtained for other indications also for determining bone density. For Hounsfield units (HU) measured with CT in specific regions of interests (ROIs) in one or several slice/s a correlation with bone mineral density (BMD) measured by DEXA in humans and dogs of between 0.44 and 0.77 is reported in the literature. In the present study, instead certain volumes of interest (VOIs) obtained by CT scan and the corresponding HU to the respective VOIs were compared with the bone mineral density of the corresponding areas measured by DEXA. The aim of the study was to investigate whether this procedure gives more accurate information about bone density of the bones as three-dimensional objects of the respective patient. Correlation between measured HU in the respective VOI and BMD measured with DEXA in the corresponding ROI showed a very good correlation of 0.93. Linear regression with R2 = 0.85 (p = 0.0262) was calculated. Except for VOI5, similar distribution of values and significant differences (p < 0.0001-0.0087) between ROIs/VOIs were detected. Determining HU for assessing bone mineral density in a certain volume provides more accurate results than those previously reported from two-dimensional (2D) CT measurements. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2667-2672, 2017.

Comparative kinematic gait analysis in young and old Beagle dogs.

Age-related involution in dogs involves loss of muscle mass and changes in connective tissue and articular cartilage. The aim of this study was to examine whether an age-related influence on joint mobility can be detected in the absence of disease. Five young (mean age 2.0 years) and five old (mean age 10.4 years) healthy and sound Beagle dogs underwent computer-assisted gait analysis during locomotion on a treadmill. Shoulder, elbow, carpal, hip, stifle, and tarsal joint angles including joint angle progression curves, minimum and maximum joint angles, and range of motion (ROM) in degrees were analyzed. The old group had a smaller maximum joint angle (p = 0.037) and ROM (p = 0.037) of the carpal joint; there were similar tendencies in the shoulder, elbow, and carpal joints. Descriptive analysis of the progression curves revealed less flexion and extension of the forelimb joints. The results indicate restricted joint mobility of the forelimb in old dogs, primarily of the carpal joint. Results in the joints of the hindlimb were inconsistent, and the contrasting alterations may be due to a compensatory mechanism. As most alterations were found in the distal joints, these should receive particular attention when examining elderly dogs.

Radiographic evaluation of early periprosthetic acetabular bone contrast and prosthetic head acetabular coverage after uncemented and cemented total hip prosthesis in dogs.

BACKGROUND: Coxofemoral osteoarthritis is a chronic, disabling condition affecting people and dogs, with THA providing an excellent return to function in severely affected joints. The principal role of THA is to restore an adequate range of motion to the hip joint while transferring load from the acetabulum in order to improve the survival of the implant and enhance the limb function in the short and long terms. The objectives of the study reported here were, therefore, to radiographically evaluate periprosthetic acetabular bone GV and to assess prosthetic head acetabular coverage after 4 months of uncemented and cemented THA in dogs. Means periprosthetic acetabular GV for each and combined 3 regions of interest (zones 1, 2 and 3) were calculated immediately and 4 months after THA. Prosthetic head Norberg (PHN) angle was also measured to assess the degree of prosthetic head acetabular coverage after 4 months of surgery. RESULTS: Zones 2 and 3 showed a significant increase in the mean bone GV after 4 months of uncemented THA. No differences in zones 1-3 after 4 months of cemented THA. Combined zones showed a significant increase in overall mean bone GV 4 months after uncemented THA; whereas, no changes were identified after 4 months of cemented THA. The PHN angles did not change after 4 months of uncemented and cemented THA and did not differ significantly between the 2 designs of hip arthroplasty. CONCLUSIONS: Regional periprosthetic acetabular bone GV varies with the design of THA. None of the designs showed periprosthetic acetabular bone lucency. No differences identified in the degree of prosthetic head acetabular coverage in both designs, indicating proper implant stability after 4 months of surgery. Further longer-term investigation on larger population is however still warranted.

Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies.

Screening for pharmaceutically viable stability from measurements of thermally induced protein unfolding and short-term accelerated stress underpins much molecule design, selection, and formulation in the pharmaceutical biotechnology industry. However, the interrelationships among intrinsic protein conformational stability, thermal denaturation, and pharmaceutical stability are complex. There are few publications in which predictions from thermal unfolding-based screening methods are examined together with pharmaceutically relevant long-term storage stability performance. We have studied eight developable therapeutic IgG molecules under solution conditions optimized for large-scale commercial production and delivery. Thermal unfolding profiles were characterized by differential scanning calorimetry (DSC) and intrinsic fluorescence recorded simultaneously with static light scattering (SLS). These molecules exhibit a variety of thermal unfolding profiles under common reference buffer conditions and under individually optimized formulation conditions. Aggregation profiles by SE-HPLC and bioactivity upon long-term storage at 5, 25, and 40 °C establish that IgG molecules possessing a relatively wide range of conformational stabilities and thermal unfolding profiles can be formulated to achieve pharmaceutically stable drug products. Our data suggest that a formulation design strategy that increases the thermal unfolding temperature of the Fab transition may be a better general approach to improving pharmaceutical storage stability than one focused on increasing Tonset or Tm of the first unfolding transition.

Evaluation of thoracic limb loads, elbow movement, and morphology in dogs before and after arthroscopic management of unilateral medial coronoid process disease.

OBJECTIVE: To (1) evaluate thoracic limb loads and symmetry, and elbow function and morphology, before and after arthroscopic treatment of unilateral medial coronoid process disease (MCPD), and (2) determine if functional variables correlate with morphologic findings. STUDY DESIGN: Prospective case series. ANIMALS: Dogs (n = 14) with thoracic limb lameness. METHODS: Dogs were included when unilateral MCPD was confirmed as the cause of lameness. Kinetic analysis of both thoracic limbs, along with kinematic analysis and goniometry of both elbows were carried out before, and 60, 120, and 180 days after partial coronoidectomy by arthroscopy. Radiography and computed tomography of both elbows were performed before and 180 days after arthroscopy. RESULTS: A nonsignificant (P = .11) increase in the peak vertical loads (PFz), and a significant (P = .022) increase in the vertical impulse (iFz) applied by the affected limb were seen. Symmetry indices improved, with significant differences between sessions (PFz: P = .019; iFz: P = .003). Kinematic variables showed no significant differences, between sessions or when comparing both elbows within sessions. Goniometry revealed no significant differences between sessions, but some significant differences were identified when comparing both elbows within sessions. Osteophytosis and degree of lameness showed no correlation, before (rs = -0.077; P = .79) or after arthroscopy (rs = 0.27; P = .35). CONCLUSIONS: Kinetic variables improved after arthroscopy, without full restoration of function. Kinematic variables did not change significantly. Osteoarthritis and goniometric measurements in the affected joint worsened. Functional variables did not correlate with morphologic findings.

Group IV cytosolic phospholipase A2 mediates arachidonic acid release in H9c2 rat cardiomyocyte cells in response to hydrogen peroxide.

Damaging reactive oxygen species are released during episodes of ischemia and reperfusion. Some cellular adaptive responses are triggered to protect the injured organ, while other cascades are triggered which potentiate the damage. In these studies, we demonstrate that rat cardiomyocte H9c2 cells release arachidonic acid in response to hydrogen peroxide. In H9c2 cells, arachidonic acid release is attenuated by methyl arachidonyl fluorophosphonate (MAFP) and pyrrophenone, indicating that a phospholipase A2 Group IV enzyme mediates arachidonic acid mobilization. Moreover, hydrogen peroxide alters the cellular morphology of the H9c2 cells, causing drastic cell shrinkage. Because MAFP and pyrrophenone prevent the morphological alterations caused by hydrogen peroxide, these studies show that phospholipase A2 Group IV activity is likely integrally involved in the damage initiated by hydrogen peroxide.

PGE2 release is independent of upregulation of Group V phospholipase A2 during long-term stimulation of P388D1 cells with LPS.

P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.

Distinguishing phospholipase A2 types in biological samples by employing group-specific assays in the presence of inhibitors.

This manuscript reviews and updates radiolabel-based enzyme assays designed to distinguish the activity of phospholipase A2 (PLA2) types in biological samples. This approach should be useful in lipidomics studies. The assays were originally designed to differentiate between Group IVA cytosolic PLA2 (GIVA cPLA2), Group VIA calcium-independent PLA2 (GVIA iPLA2), Group IIA secreted PLA2 (GIIA sPLA2) and Group V secreted PLA2 (GV sPLA2). The specificity of these assays has now been confirmed using purified, recombinant human PLA2s and the utility of these assays is demonstrated with rat spinal cord homogenate as an example of a biological tissue sample of interest to the neuroscience community. Modifications to the original assays by the addition of group-specific inhibitors are presented to ensure the specificity of the assays and to further differentiate between recently identified PLA2s. Specific tests are suggested to confirm the specificity of each assay. Additionally, it was discovered that one commonly used GIVA cPLA2/GVIA iPLA2 inhibitor, methyl arachidonyl fluorophosphonate (MAFP) from one commercial source, was found to inhibit GIIA sPLA2 and GV sPLA2, but not GIVA cPLA2, presumably due to oxidation of the compound during shipment, resulting in a different molecule with altered specificity.

Spinal phospholipase A2 in inflammatory hyperalgesia: role of group IVA cPLA2.

Current work has shown the importance of spinal cyclooxygenase (COX) products in facilitatory processes leading to tissue injury induced hyperalgesia. This cascade must originate with free arachidonic acid (AA) released by the activity of spinal phospholipase A2s (PLA2). In the present work, we studied the role of PLA2s in spinal sensitization. We first demonstrate the presence of constitutive mRNA in the spinal cord for PLA2 Groups IB, IIA, IIC, IVA, V and VI by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Using quantitative-PCR, we found that Group IVA cPLA2 and Group VI iPLA2 are the predominant PLA2 messages in the spinal cord. Western blotting and activity assays specific for Group IVA cPLA2 and Group VI iPLA2 verified the presence of these enzymes. PLA2 activity in spinal cord homogenates was suppressed by methyl arachidonyl fluorophosphonate (MAFP) and arachidonyl trifluoromethylketone (AACOCF3), mixed inhibitors of Group IVA cPLA2 and Group VI iPLA2 as well as by bromoenol lactone (BEL), a Group VI iPLA2 inhibitor. The spinal expression of PLA2 mRNA or protein was not altered in the face of peripheral inflammation. Secondly, we showed that intrathecal (i.t.) administration of MAFP and AACOCF3, but not BEL, dose-dependently prevented thermal hyperalgesia induced by intraplantar carrageenan as well as formalin-induced flinching. Finally, i.t. injection of AACOCF3, at antihyperalgesic doses, decreased the release of prostaglandin E2 (PGE2) into spinal dialysate evoked by i.t. NMDA, while i.t. injection of BEL had no effect. Taken together, this work points to a role for constitutive Group IVA cPLA2 in spinal nociceptive processing.

The ABC's of Group IV cytosolic phospholipase A2.

The three known human Group IV phospholipase A2 (PLA2) paralogs, Group IVA, IVB and IVC, were overexpressed in Sf9 insect cells using the baculovirus expression system. An endogenous, calcium-independent PLA2 activity was identified in the insect cell lysates, which can be inhibited by bromoenol lactone (BEL). The Group IV PLA2 enzymes were characterized in overexpressing insect cell lysates in the presence of BEL, enabling their differentiation from the endogenous PLA2 activity. Group IVC PLA2 was found to have significant lysophospholipase activity, while Group IVB PLA2 did not. Of the three paralogs, only the Group IVA PLA2 shows enhanced activity in the presence of PIP2, which enables its differential detection in cell homogenates. RT-PCR was used to demonstrate the presence of all three enzymes in human U937 and human WISH cells, while only Group IVA and Group IVB PLA2 were detected in murine P388D1 cells and human astrocytes at the mRNA level.

Expression and function of phospholipase A(2) in brain.

Phospholipase A(2) (PLA(2)) appears to play a fundamental role in cell injury in the central nervous system. We have investigated PLA(2) expression in the astrocytoma cell line 1231N1, and found that GIVA, GIVB, GIVC and GVI PLA(2) messages are expressed. PLA(2) activity is increased by inflammatory/injury stimuli such as interleukin-1beta and lipopolysaccharide in these cells but with very different time courses. The arachidonic acid liberated is converted to prostaglandin E(2), possibly by cyclooxygenase-2, which is induced by inflammatory stimuli. This cell system emerges as a model to study injury/inflammation-related activation of the new PLA(2) forms GIVB and GIVC.