Intra-amniotic rAAV-mediated microdystrophin gene transfer improves canine X-linked muscular dystrophy and may induce immune tolerance.

Duchenne muscular dystrophy (DMD) is a severe congenital disease due to mutations in the dystrophin gene. Supplementation of dystrophin using recombinant adenoassociated virus vector has promise as a treatment of DMD, although therapeutic benefit of the truncated dystrophin still remains to be elucidated. Besides, host immune responses against the vector as well as transgene products have been denoted in the clinical gene therapy studies. Here, we transduced dystrophic dogs fetuses to investigate the therapeutic effects of an AAV vector expressing microdystrophin under conditions of immune tolerance. rAAV-CMV-microdystrophin and a rAAV-CAG-luciferase were injected into the amniotic fluid surrounding fetuses. We also reinjected rAAV9-CMV-microdystrophin into the jugular vein of an infant dystrophic dog to induce systemic expression of microdystrophin. Gait and cardiac function significantly improved in the rAAV-microdystrophin-injected dystrophic dog, suggesting that an adequate treatment of rAAV-microdystrophin with immune modulation induces successful long-term transgene expression to analyze improved dystrophic phenotype.

Staphylococcus pseudintermedius ST2660 isolated from a cat has strong biofilm-forming ability and increases biofilm formation at cat's normal body temperature.

Staphylococcus pseudintermedius has been isolated from dogs, cats, and horses and is also known as an emergent zoonotic agent. We administered orbifloxacin, a fluoroquinolone, to treat bacterial infections of cutaneous wounds caused by excessive grooming of the skin in contact with the subcutaneous port of the subcutaneous ureteral bypass (SUB) system in a cat. However, after 80 days of treatment, a severe abscess was observed in the wound and fluoroquinolone-resistant S. pseudintermedius was isolated from the abscess. The isolate was identified as a novel sequence type (ST) 2660 and contained genes for leukocidins (lukS and lukF), exfoliative toxin (siet), and biofilm regulation (icaA and icaD). The isolate was resistant to macrolide, lincosamide, fluoroquinolone, and tetracycline classes. In addition, the isolate had strong biofilm-forming ability which significantly increased with culturing at 39 °C compared with that at 37 °C, suggesting that the isolate prefers a cats' body temperature as the optimal biofilm growth condition. Notably, the biofilms were increased in the presence of doxycycline with culturing at 39 °C. This study is the first report in Japan on the new sequence type of S. pseudintermedius isolated from a companion animal and clarifies the distinctive virulence of S. pseudintermedius.

Assessment of left ventricular regional function in affected and carrier dogs with Duchenne muscular dystrophy using speckle tracking echocardiography.

BACKGROUND: Two-dimensional speckle tracking echocardiography (STE) is a relatively new method to detect regional myocardial dysfunction. To assess left ventricular (LV) regional myocardial dysfunction using STE in Duchenne muscular dystrophy model dogs (CXMD(J)) without overt clinical signs of heart failure. METHODS: Six affected dogs, 8 carrier dogs with CXMD(J), and 8 control dogs were used. Conventional echocardiography, systolic and diastolic function by Doppler echocardiography, tissue Doppler imaging (TDI), and strain indices using STE, were assessed and compared among the 3 groups. RESULTS: Significant differences were seen in body weight, transmitral E wave and E' wave derived from TDI among the 3 groups. Although no significant difference was observed in any global strain indices, in segmental analysis, the peak radial strain rate during early diastole in posterior segment at chordae the tendineae level showed significant differences among the 3 groups. CONCLUSIONS: The myocardial strain rate by STE served to detect the impaired cardiac diastolic function in CXMD(J) without any obvious LV dilation or clinical signs. The radial strain rate may be a useful parameter to detect early myocardial impairment in CXMD(J).

Cardiac involvement in Beagle-based canine X-linked muscular dystrophy in Japan (CXMDJ): electrocardiographic, echocardiographic, and morphologic studies.

BACKGROUND: Cardiac mortality in Duchenne muscular dystrophy (DMD) has recently become important, because risk of respiratory failure has been reduced due to widespread use of the respirator. The cardiac involvement is characterized by distinctive electrocardiographic abnormalities or dilated cardiomyopathy, but the pathogenesis has remained obscure. In research on DMD, Golden retriever-based muscular dystrophy (GRMD) has attracted much attention as an animal model because it resembles DMD, but GRMD is very difficult to maintain because of their severe phenotypes. We therefore established a line of dogs with Beagle-based canine X-linked muscular dystrophy in Japan (CXMDJ) and examined the cardiac involvement. METHODS: The cardiac phenotypes of eight CXMDJ and four normal male dogs 2 to 21 months of age were evaluated using electrocardiography, echocardiography, and histopathological examinations. RESULTS: Increases in the heart rate and decreases in PQ interval compared to a normal littermate were detected in two littermate CXMDJ dogs at 15 months of age or older. Distinct deep Q-waves and increase in Q/R ratios in leads II, III, and aVF were detected by 6-7 months of age in all CXMDJ dogs. In the echocardiogram, one of eight of CXMDJ dogs showed a hyperechoic lesion in the left ventricular posterior wall at 5 months of age, but the rest had not by 6-7 months of age. The left ventricular function in the echocardiogram indicated no abnormality in all CXMDJ dogs by 6-7 months of age. Histopathology revealed myocardial fibrosis, especially in the left ventricular posterobasal wall, in three of eight CXMDJ dogs by 21 months of age. CONCLUSION: Cardiac involvement in CXMDJ dogs is milder and has slower progression than that described in GRMD dogs. The distinct deep Q-waves have been ascribed to myocardial fibrosis in the posterobasal region of the left ventricle, but our data showed that they precede the lesion on echocardiogram and histopathology. These findings imply that studies of CXMDJ may reveal not only another causative mechanism of the deep Q-waves but also more information on the pathogenesis in the dystrophin-deficient heart.

Initial pulmonary respiration causes massive diaphragm damage and hyper-CKemia in Duchenne muscular dystrophy dog.

The molecular mechanism of muscle degeneration in a lethal muscle disorder Duchene muscular dystrophy (DMD) has not been fully elucidated. The dystrophic dog, a model of DMD, shows a high mortality rate with a marked increase in serum creatine kinase (CK) levels in the neonatal period. By measuring serum CK levels in cord and venous blood, we found initial pulmonary respiration resulted in massive diaphragm damage in the neonates and thereby lead to the high serum CK levels. Furthermore, molecular biological techniques revealed that osteopontin was prominently upregulated in the dystrophic diaphragm prior to the respiration, and that immediate-early genes (c-fos and egr-1) and inflammation/immune response genes (IL-6, IL-8, COX-2, and selectin E) were distinctly overexpressed after the damage by the respiration. Hence, we segregated dystrophic phases at the molecular level before and after mechanical damage. These molecules could be biomarkers of muscle damage and potential targets in pharmaceutical therapies.