D. Michael Tillson

Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy

Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.

Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

Diagnosis and treatment of portosystemic shunts in the cat.

Feline portosystemic shunts (PSSs) are best managed with surgical intervention. To accomplish this task, veterinarians must be able to identify cats in which a PSS is a strong possibility. Cats exhibit clinical signs that are both similar to and different from the signs of PSSs in dogs. Options for imaging feline PSSs include ultrasound, scintigraphy, and contrast radiography. Medical management stabilizes the critical patients in anticipation of surgery and is used for those patients in which surgical correction is not possible. Surgical options for PSS occlusion include techniques for acute vessel ligation or attenuation and for slow vessel occlusion. The prognosis is based on the degree of shunt occlusion and the ability of the liver to adapt to increased hepatic blood flow.

Isometric responses of isolated intrapulmonary bronchioles from cats with and without adult heartworm infection.

OBJECTIVE To determine the isometric responses of isolated intrapulmonary bronchioles from cats with and without adult heartworm infection. ANIMALS 13 purpose-bred adult cats. PROCEDURES Cats were infected with 100 third-stage larvae or received a sham inoculation, and the left caudal lung lobe was collected 278 to 299 days after infection. Isometric responses of intrapulmonary bronchiolar rings were studied by use of a wire myograph. Three cycles of contractions induced by administration of 10 μM acetylcholine were followed by administration of the contractile agonists acetylcholine, histamine, and 5-hydroxy-tryptamine. To evaluate relaxation, intrapulmonary bronchiolar rings were constricted by administration of 10 μM 5-hydroxytryptamine, and concentration-response curves were generated from administration of sodium nitroprusside, isoproterenol, and substance P. RESULTS Compared with tissues from control cats, contractile responses to acetylcholine and 5-hydroxytryptamine were reduced in tissues from heartworm-infected cats. Relaxation to isoproterenol was significantly reduced in tissues from heartworm-infected cats. Relaxation to substance P was increased in tissues from heartworm-infected cats, but relaxation to sodium nitroprusside was unchanged. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that despite increased bronchiolar wall thickness in heartworm-infected cats, a hyperreactive response of the bronchiolar smooth muscle is not the primary mechanism of respiratory tract clinical signs. Reduced response of the airway to isoproterenol may indicate refractoriness to bronchiolar relaxation in heartworm-infected cats.

Effect of Vascular Seal Configuration Using the LigaSure on Arterial Challenge Pressure, Time for Seal Creation, and Histologic Features

Objective To determine the effectiveness of a vascular sealing technology on canine carotid arteries using various seal configurations to achieve maximal vessel security. Study Design Ex-vivo study. Animals Dogs (n = 20). Methods Carotid arteries (n = 40) were removed from the mid-cervical region of recently euthanatized dogs. Harvested vessels were closed with 2 circumferential ligatures (Group 1) or a vascular sealing device using 1 of 4 seal configurations of 1 or 2 seals combined with 1 or 2 machine activations/seal. The artery was instrumented to measure intraluminal pressure to evaluate the security of each seal during saline infusion. Maximum intraluminal pressure was recorded for each group, and time for application of each sealing protocol was compared using 1-way ANOVA and Tukeys test for multiple comparisons. Histologic features of the sealing protocols were evaluated. Results Arterial closures for each group were effective in preventing leakage up to 300 mmHg. There was no significant difference in maximum intraluminal pressure between any group. A significant difference (P ≤ .001) was observed for time to seal creation between the groups using 1 and 2 seals. Histologic evaluation showed no differences between the different sealing protocols. Conclusion Vessel sealing using a single seal created with a single activation cycle was adequate for sealing canine carotid arteries. Histologic examination did not demonstrate any disadvantages to multiple seals or multiple cycle activations.OBJECTIVE To determine the effectiveness of a vascular sealing technology on canine carotid arteries using various seal configurations to achieve maximal vessel security. STUDY DESIGN Ex-vivo study. ANIMALS Dogs (n = 20). METHODS Carotid arteries (n = 40) were removed from the mid-cervical region of recently euthanatized dogs. Harvested vessels were closed with 2 circumferential ligatures (Group 1) or a vascular sealing device using 1 of 4 seal configurations of 1 or 2 seals combined with 1 or 2 machine activations/seal. The artery was instrumented to measure intraluminal pressure to evaluate the security of each seal during saline infusion. Maximum intraluminal pressure was recorded for each group, and time for application of each sealing protocol was compared using 1-way ANOVA and Tukeys test for multiple comparisons. Histologic features of the sealing protocols were evaluated. RESULTS Arterial closures for each group were effective in preventing leakage up to 300 mmHg. There was no significant difference in maximum intraluminal pressure between any group. A significant difference (P ≤ .001) was observed for time to seal creation between the groups using 1 and 2 seals. Histologic evaluation showed no differences between the different sealing protocols. CONCLUSION Vessel sealing using a single seal created with a single activation cycle was adequate for sealing canine carotid arteries. Histologic examination did not demonstrate any disadvantages to multiple seals or multiple cycle activations.

Thoracic Surgery; Important Considerations and Practical Steps

Thoracic surgery is a challenge for any veterinary surgeon. A review of several important articles on topics relative to thoracotomy procedures is presented. Discussion also includes an evaluation of availability of appropriate surgical facilities, necessary equipment before undertaking thoracic surgical procedures, and the essentials and pitfalls to making an approach and effectively closing the thoracic cavity of a dog or cat. This article reviews the 3 primary types of thoracotomy: lateral (intercostal) thoracotomy, median sternotomy, and transdiaphragmatic thoracotomy. Essential anatomy, surgical approach, and various techniques to ensure effective and durable thoracotomy closure are presented.

High-resolution computed tomography evaluation of the bronchial lumen to vertebral body diameter and pulmonary artery to vertebral body diameter ratios in anesthetized ventilated normal cats

Objectives Bronchial lumen to pulmonary artery diameter (BA) ratio has been utilized to investigate pulmonary pathology on high-resolution CT images. Diseases affecting both the bronchi and pulmonary arteries render the BA ratio less useful. The purpose of the study was to establish bronchial lumen diameter to vertebral body diameter (BV) and pulmonary artery diameter to vertebral body diameter (AV) ratios in normal cats. Methods Using high-resolution CT images, 16 sets of measurements (sixth thoracic vertebral body [mid-body], each lobar bronchi and companion pulmonary artery diameter) were acquired from young adult female cats and 41 sets from pubertal female cats. Results Young adult and pubertal cat BV ratios were not statistically different from each other in any lung lobe. Significant differences between individual lung lobe BV ratios were noted on combined age group analysis. Caudal lung lobe AV ratios were significantly different between young adult and pubertal cats. All other lung lobe AV ratios were not significantly different. Caudal lung lobe AV ratios were significantly different from all other lung lobes but not from each other in both the young adult and pubertal cats. Conclusions and relevance BV ratio reference intervals determined for individual lung lobes could be applied to both young adult and pubertal cats. Separate AV ratios for individual lung lobes would be required for young adult and pubertal cats. These ratios should allow more accurate evaluation of cats with concurrent bronchial and pulmonary arterial disease.

Radiographic evaluation of subcutaneously injected, water-soluble, iodinated contrast for lymphography in dogs

Sentinel lymph node (SLN) mapping is common in many types of human cancers, and is gaining utility in veterinary medicine. There are currently many different methods described in veterinary medicine for pre-operative SLN mapping, however, most of these are restricted to referral institutions due to cost and need for specialized equipment. The purpose of this prospective, pilot study was to evaluate the feasibility of radiographic evaluation of water-soluble, iodinated contrast (WIC) injected subcutaneously for lymphography in dogs. Eight dogs were injected with 1-2 milliliters of WIC into the subcutaneous tissues overlying the tarsus in 4 separate locations mimicking a circumferential, peri-tumoral injection. Radiographs were taken at select time points up to 50 minutes. Image sequences were evaluated by a single, board-certified radiologist. All 8 dogs had visible contrast-enhancing lymphatic channels. Median time to lymphatic enhancement was immediately post-injection. Seven dogs (88%) had 8 contrast enhancing lymph nodes (7 popliteal and 1 superficial inguinal). Median time to lymph node enhancement was 20 minutes. In this study, the plantar aspect of the pes drained to the superficial inguinal lymph node, and the dorsal aspect of the pes drained to the popliteal lymph node. Subcutaneously-injected WIC was readily identifiable in the lymphatic channels and draining lymph node(s). Subcutaneously injected WIC may offer a practical alternative to previously described pre-operative methods of SLN mapping. Additionally, one cannot assume that the popliteal lymph node alone, drains the distal pelvic limb.

High-resolution CT evaluation of bronchial lumen to vertebral body, pulmonary artery to vertebral body and bronchial lumen to pulmonary artery ratios in Dirofilaria immitis-infected cats with and without selamectin administration

Objectives The bronchial lumen to pulmonary artery (BA) ratio is utilized to evaluate pulmonary pathology on CT images. The BA ratio may be unreliable when changes are present in bronchial and pulmonary arteries concurrently. Bronchial lumen to vertebral body (BV) and pulmonary artery to vertebral body (AV) ratios have been established in normal cats and may serve as an alternative. This study aimed to evaluate the BV, AV and BA ratios in cats before and after infection with Dirofilaria immitis, with and without selamectin administration, and to characterize the distribution of disease. Methods Archived CT images were reviewed from three groups of cats: D immitis-infected untreated (n = 6); infected pretreated with selamectin (n = 6); and uninfected untreated (n = 5). The BV, AV and BA ratios were calculated for all lung lobes for baseline (D0) and day 240 (D240) postinfection. Ratios and percentage change from baseline were compared between lobes and between groups. Results BV and AV ratios were more consistent in identifying abnormalities when disease was present in bronchial and arteries concurrently than BA ratios. Infected untreated cats had significant changes in both BV and AV ratios and percentage change from baseline. Abnormal BV and AV ratios were noted in the infected selamectin group, although less widely distributed. Conclusions and relevance The BV and AV ratios more accurately identified bronchial and pulmonary artery abnormalities in D immitis-infected cats. Both bronchial and pulmonary artery changes were present in infected cats, decreasing the diagnostic application of the BA ratio. Pulmonary artery changes were more widely distributed than bronchial changes in the lung. Heartworm-infected cats receiving selamectin had bronchial and pulmonary artery changes but to a lesser extent than untreated heartworm-infected cats. The CT-derived BV and AV ratios are a useful measure to evaluate lung disease of cats.

Effect of Vascular Seal Configuration Using the LigaSure on Arterial Challenge Pressure, Time for Seal Creation, and Histologic Features: LigaSure Vascular Seal Characteristics

Objective To determine the effectiveness of a vascular sealing technology on canine carotid arteries using various seal configurations to achieve maximal vessel security. Study Design Ex-vivo study. Animals Dogs (n = 20). Methods Carotid arteries (n = 40) were removed from the mid-cervical region of recently euthanatized dogs. Harvested vessels were closed with 2 circumferential ligatures (Group 1) or a vascular sealing device using 1 of 4 seal configurations of 1 or 2 seals combined with 1 or 2 machine activations/seal. The artery was instrumented to measure intraluminal pressure to evaluate the security of each seal during saline infusion. Maximum intraluminal pressure was recorded for each group, and time for application of each sealing protocol was compared using 1-way ANOVA and Tukeys test for multiple comparisons. Histologic features of the sealing protocols were evaluated. Results Arterial closures for each group were effective in preventing leakage up to 300 mmHg. There was no significant difference in maximum intraluminal pressure between any group. A significant difference (P ≤ .001) was observed for time to seal creation between the groups using 1 and 2 seals. Histologic evaluation showed no differences between the different sealing protocols. Conclusion Vessel sealing using a single seal created with a single activation cycle was adequate for sealing canine carotid arteries. Histologic examination did not demonstrate any disadvantages to multiple seals or multiple cycle activations.OBJECTIVE To determine the effectiveness of a vascular sealing technology on canine carotid arteries using various seal configurations to achieve maximal vessel security. STUDY DESIGN Ex-vivo study. ANIMALS Dogs (n = 20). METHODS Carotid arteries (n = 40) were removed from the mid-cervical region of recently euthanatized dogs. Harvested vessels were closed with 2 circumferential ligatures (Group 1) or a vascular sealing device using 1 of 4 seal configurations of 1 or 2 seals combined with 1 or 2 machine activations/seal. The artery was instrumented to measure intraluminal pressure to evaluate the security of each seal during saline infusion. Maximum intraluminal pressure was recorded for each group, and time for application of each sealing protocol was compared using 1-way ANOVA and Tukeys test for multiple comparisons. Histologic features of the sealing protocols were evaluated. RESULTS Arterial closures for each group were effective in preventing leakage up to 300 mmHg. There was no significant difference in maximum intraluminal pressure between any group. A significant difference (P ≤ .001) was observed for time to seal creation between the groups using 1 and 2 seals. Histologic evaluation showed no differences between the different sealing protocols. CONCLUSION Vessel sealing using a single seal created with a single activation cycle was adequate for sealing canine carotid arteries. Histologic examination did not demonstrate any disadvantages to multiple seals or multiple cycle activations.