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J N Kornegay

Publications and source records attributed to J N Kornegay.

At least 19 recordsLinked to original sources
PubMed2012

MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were administered with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with magnetic resonance imaging (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30&#xb1;10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a 3-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.

Animals↗
PubMed2001

Myofiber injury and regeneration in a canine homologue of Duchenne muscular dystrophy.

OBJECTIVE: To test the hypothesis that differential skeletal muscle involvement, previously observed in dogs with a homologue of Duchenne muscular dystrophy, correlates with the histochemical markers of myofiber injury and regeneration. DESIGN: Evidence of injury (cellular penetration by Evans blue dye, immunoglobulin G expression, hematoxylin and eosin staining of necrotic figures), myofiber regeneration (fetal myosin heavy chain isoform expression), and morphologic indices in the cranial sartorius (CS), long digital extensor, and vastus lateralis muscles were examined in five dogs with dystrophy and five normal dogs. RESULTS: Only the CS muscle, at 1 mo, demonstrated significant differences in injury when compared with age-matched controls. By 6 mo, the long digital extensor and vastus lateralis also suffered greater than normal injury. Only the dystrophic CS tissue expressed a notable increase in mean myofiber diameter when compared with other muscles at 6 mo. Normal CS muscles revealed a distinct population of small myofibers at this age. CONCLUSION: The CS seems unique in its selective pathologic involvement. These differences may contribute to the marked regenerative response of this muscle in the dystrophic state. An improved understanding of mechanisms by which some dystrophin-deficient canine muscles remain spared from injury may provide clues to investigate and prevent the degenerative processes in humans.

Animals↗
PubMed2000

In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide.

In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric oligonucleotide to induce host cell mismatch repair mechanisms and correct the chromosomal mutation to wild type. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated in vivo repair of the GRMD mutation that was sustained for 48 weeks. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of exons 5-10 demonstrated increasing levels of exon 7 inclusion with time. An isolated exon 7-specific dystrophin antibody confirmed synthesis of normal-sized dystrophin product and positive localization to the sarcolemma. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism (RFLP)-PCR and sequencing the PCR product. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscle of a live animal using a chimeric oligonucleotide.

Animals↗
PubMed1999

Contraction force generated by tarsal joint flexion and extension in dogs with golden retriever muscular dystrophy.

Force generated due to torque caused by tarsal joint flexion and extension was measured noninvasively at 3, 4.5, 6, and 12 months of age in dogs with the Duchenne homologue, golden retriever muscular dystrophy (GRMD). Absolute and body-weight-corrected GRMD twitch and tetanic force values were lower than normal at all ages (P<0.01 for most). Tarsal flexion and extension were differentially affected. Flexion values were especially low at 3 months, whereas extension was affected more at later ages. Several other GRMD findings differed from normal. The twitch/tetany ratio was generally lower; post-tetanic potentiation for flexion values was less marked; and extension relaxation and contraction times were longer. The consistency of GRMD values was studied to determine which measurements will be most useful in evaluating treatment outcome. Standard deviation was proportionally greater for GRMD versus normal recordings. More consistent values were seen for tetany versus twitch and for flexion versus extension. Left and right limb tetanic flexion values did not differ in GRMD; extension values were more variable. These results suggest that measurement of tarsal tetanic force should be most useful to document therapeutic benefit in GRMD dogs.

Age Factors↗
PubMed1999

Molecular analysis of a spontaneous dystrophin 'knockout' dog.

We have determined the molecular basis for skeletal myopathy and dilated cardiomyopathy in two male German short-haired pointer (GSHP) littermates. Analysis of skeletal muscle demonstrated a complete absence of dystrophin on Western blot analysis. PCR analysis of genomic DNA revealed a deletion encompassing the entire dystrophin gene. Molecular cytogenetic analysis of lymphocytes from the dam and both dystrophic pups confirmed a visible deletion in the p21 region of the affected canine X chromosome. Utrophin is up-regulated in the skeletal muscle, but does not appear to ameliorate the dystrophic canine phenotype. This new canine model should further our understanding of the physiological and biochemical processes in Duchenne muscular dystrophy.

Animals↗
PubMed1999

Hydranencephaly and cerebellar hypoplasia in two kittens attributed to intrauterine parvovirus infection.

Six weeks after vaccination with modified live feline parvovirus vaccine, a cat gave birth to five kittens, three of which died soon afterwards. The remaining two kittens (A and B) survived, but at 8 weeks of age were unable to walk and showed abnormal behaviour, with lack of menace and oculovestibular responses, and severe dysmetria. These signs suggested multifocal disease associated with the cerebrum and cerebellum. Magnetic resonance imaging demonstrated severe bilateral (kitten A) or unilateral (kitten B) hydrocephalus or hydranencephaly, combined with cerebellar agenesis (kitten A) or severe hypoplasia (kitten B). Hydranencephaly was confirmed histopathologically in both kittens. Parvovirus was isolated from the kidney of one kitten. Parvoviral DNA was amplified by the polymerase chain reaction (PCR) from paraffin wax-embedded brain of both kittens. The severe malformations observed in these kittens presumably resulted from an in-utero parvovirus infection, possibly due to vaccination, that occurred late in the first, or early in the second, trimester of pregnancy.

Animals↗
PubMed1998

Evaluating motor end-plate-targeted injections of botulinum toxin type A in a canine model.

Tarsal joint forces were measured in dogs over 70 days following botulinum toxin type A (BTX-A) injections. Three dogs were injected at motor end-plates located by electromyography (EMG), while 3 dogs were similarly injected, but without EMG guidance. Extension forces were significantly (P < 0.05) smaller in limbs injected at motor end-plates than in corresponding limbs on days 14 and 35. There were no significant differences at other times. Using these techniques, EMG end-plate targeting potentiates effects of BTX-A.

Animals↗
PubMed1998

Alternative dystrophin gene transcripts in golden retriever muscular dystrophy.

Golden retriever muscular dystrophy (GRMD), the canine model of Duchenne muscular dystrophy (DMD), is caused by a splice site mutation in the dystrophin gene. This mutation predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. Western blot analysis of skeletal muscle from GRMD dogs reveals a slightly truncated 390-kD protein that is approximately 91% the size of normal dystrophin. This 390-kD dystrophin suggests that GRMD dogs, like some DMD patients, employ a mechanism to overcome their predicted frameshift. Reverse-transcriptase polymerase chain reaction on GRMD muscle has revealed two in-frame dystrophin transcripts which lack either exons 3-9 or exons 5-12. Both transcripts could be translated into a dystrophin protein of approximately 390 kD. An understanding of how truncated dystrophin is produced in GRMD may allow this mechanism to be manipulated toward a potential therapy for DMD.

Alternative Splicing↗
PubMed1998

Late-onset progressive spinocerebellar degeneration in Brittany Spaniel dogs.

Eight Brittany Spaniel dogs, seven females and one male, between 7 and 14 years old presented with clinical neurological signs of spinocerebellar disease of about 6 months to 4 years duration. Clinically the dogs had a dramatic forward "saluting" movement of the thoracic limbs, hypermetria of the pelvic limbs, cerebellar ataxia and intention tremors. Terminally, dogs crawled in a crouched thoracic posture with neck extension. Lesions were confined to cerebellum, medulla oblongata and spinal cord. The most severe lesion was diffuse Purkinje cell loss with massive neurofilament accumulation in degenerating cells. There was some bilateral neuronal degeneration in the dorsal horns of the spinal cord and in the gracilis and cuneate nuclei. There was bilateral sporadic axonal degeneration in the dorsal columns and lateral and ventromedial areas of the spinal cord. The etiology of this syndrome was not determined.

Animals↗
PubMed1997

Atraumatic rupture of the gastrocnemius muscle after corticosteroid administration in a dog.

A 6-year-old spayed female Shetland Sheepdog was referred for evaluation of lameness, muscle atrophy, and a partial plantigrade stance of the right hind limb of 5 weeks' duration. Without history of trauma, atraumatic rupture of the right gastrocnemius muscle was diagnosed. Surgical repair was unsuccessful. The dog then developed signs of hyperadrenocorticism. Results of ACTH stimulation and low-dose dexamethasone suppression tests were consistent with iatrogenic adrenal suppression. One deleterious effect of excessive use of corticosteroids on muscle and connective tissue is degenerative myopathy. Steroid-induced myopathy with subsequent rupture of the gastrocnemius muscle was suspected in this dog. Clinical signs of myopathy most often develop with use of triamcinolone acetonide; therefore, care should be taken when administering this and other corticosteroids.

Animals↗
PubMed1996

Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs.

OBJECTIVES: To use exon 7-specific genomic polymerase chain reaction (PCR) products to identify the genotypes of normal, affected, and carrier female dogs in pedigrees segregating Golden Retriever muscular dystrophy (GRMD), and to confirm the concordant segregation of the mutation in all carrier and affected dogs presently available. DESIGN: The GRMD mutation is found in the consensus splice acceptor site in intron 6 of the canine dystrophin gene. PCR cycle-sequencing and restriction fragment length polymorphism/PCR were used for determination of the pattern of segregation of the point mutation which causes GRMD. ANIMALS: Normal, clinically affected, and obligate carrier dogs in pedigrees of GRMD. PROCEDURE: DNA from blood was amplified, using PCR and primers that bracket all of exon 7 of the canine dystrophin gene as well as 100 base pairs of intron on either side. PCR products were either cycle-sequenced directly or submitted to a second round of PCR, using 1 of the original primers coupled with a mutagenic restriction fragment length polymorphism-primer, which thus creates an artificial restriction site. Digestion with Stu I detected the normal allele. To detect the affected allele, Sau96 I was used to digest the 310-base pair exon 7 genomic fragment directly. CONCLUSIONS: Simple, clear diagnosis of carrier status was possible using these methods. This mutation is passed through all carrier and affected dogs in both United States GRMD colonies and the colony in Australia. CLINICAL RELEVANCE: Rapid, accurate diagnosis of carrier and affected dogs will enhance study of this homologue of Duchenne muscular dystrophy.

Alleles↗
PubMed1994

Alpha-dystroglycan deficiency correlates with elevated serum creatine kinase and decreased muscle contraction tension in golden retriever muscular dystrophy.

The dystrophin-glycoprotein complex was examined in dystrophin-deficient dogs with golden retriever muscular dystrophy (GRMD) using immunoblot and immunofluorescence analysis. The dystrophin-associated proteins were substantially reduced in muscle from dogs with GRMD. Interestingly, regression analysis revealed a strong correlation between the amount of alpha-dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.

Animals↗
PubMed1994

A role for mast cells in the progression of Duchenne muscular dystrophy? Correlations in dystrophin-deficient humans, dogs, and mice.

Dystrophin deficiency has been shown to be the underlying cause of Duchenne muscular dystrophy. Although dystrophin-deficient homologous animal models have been identified (dog, mouse, and cat), the clinical expression of the biochemical defect is species-specific. Thus, while the genetics and biochemistry of Duchenne dystrophy is understood, the pathophysiological cascade leading to muscle weakness in only humans and dogs remains obscure. To begin to dissect the pathophysiology at the histological level, we undertook a systematic study of mast cells in normal and dystrophin-deficient muscle. Mast cells have been implicated in the development of fibrosis in other disorders, and progressive fibrosis has been hypothesized to mediate the failure of muscle regeneration in human and dog dystrophin deficiency. Our results show a strong correlation between mast cell content and localization, and the clinico-histopathological progression in humans, dogs and mice. The mast cell increases were disease specific: other dystrophic myopathies with normal dystrophin generally did not show substantial increases in mast cell content or degranulation. Our data suggest that mast cell accumulation and degranulation may cause the grouped necrosis characteristic of dystrophin deficiency in all species.

Animals↗
PubMed1994

Contraction tension and kinetics of the peroneus longus muscle in golden retriever muscular dystrophy.

Contraction tension and kinetics of the peroneus longus muscle were studied in dogs with the Duchenne homologue, golden retriever muscular dystrophy (GRMD), in advance of evaluating localized therapies such as myoblast transplantation. Absolute and both muscle- and body-weight-corrected twitch tension in GRMD dogs were low compared to normal litter mates at 3 months of age (p < 0.0005 for all). Tetanic tension was affected similarly. However, whereas absolute values were still reduced at 6 months (p < 0.0005 for twitch and 0.005 for tetany), twitch and tetanic tension corrected for either muscle or body weight was not statistically different, suggesting that the peroneus longus may be relatively spared in GRMD. Post-tetanic potentiation was more pronounced in GRMD versus normal dogs at both 3 (p < 0.0001) and 6 (p < 0.01) months. The degree of positive staircase at 3 months of age did not differ. Twitch contraction and relaxation times were dramatically prolonged, and there was concomitant sustained electrical activity, at, or before, 6 months of age in some severely affected dogs. Relatively few carriers were evaluated at these ages, but their values were similar to those of normal dogs. Apparent sparing of the peroneus longus muscle may limit application of this technique to evaluation of therapies administered early in life or in combination with toxins. Treatment to alter changes in contraction kinetics could also be assessed.

Aging↗
PubMed1994

Immunohistochemical detection of neural cell adhesion molecule and laminin in X-linked dystrophic dogs and mdx mice.

Although dystrophin deficiency is known to be the genetic and biochemical defect causing Duchenne muscular dystrophy (DMD), much remains unknown about the underlying factors affecting clinical and pathological expression of the disease. Two animal forms of muscular dystrophy resembling DMD have been described. Neural cell adhesion molecule (NCAM) and laminin expression were examined in the proliferation-competent mdx mouse and non-regenerative "golden retriever muscular dystrophy dog" (GRMD). The results showed that (1) NCAM expression was greater in dystrophic dogs and mice than in age-matched normal animals, (2) myoblast-specific NCAM was greater in mdx mice than in dystrophic dogs, and (3) laminin strongly labelled mdx and GRMD myofibre membranes but was also sometimes found in individual interstitial cells of mdx muscle. Expression of these proteins may partly determine the clinicopathological expression of dystrophin deficiency.

Animals↗
PubMed1994

Tarsal joint contracture in dogs with golden retriever muscular dystrophy.

Golden retriever muscular dystrophy (GRMD) is an X-linked myopathy homologous with Duchenne muscular dystrophy of human beings. Affected dogs have progressive clinical dysfunction due to muscle wasting and contractures. Deficits progress particularly rapidly between 3 and 6 months of age. To better characterize the role of contractures in this deterioration, the flexor surface, nonweight-bearing tarsal joint angle was measured in GRMD-affected dogs and clinically normal littermates at both ages. The mean +/- SD tarsal joint angle for clinically normal dogs decreased from 164.6 +/- 6.09 degrees to 145.6 +/- 8.80 degrees between 3 and 6 months (P < 0.0005). The value for GRMD-affected dogs decreased from 153.3 +/- 11.44 degrees to 117.6 +/- 24.55 degrees (P < 0.005). The angle for clinically normal dogs was greater than that for GRMD-affected dogs at both ages (P < 0.05 at 3 months and P < 0.01 at 6 months). The decrease in tarsal joint angle, at times to less than 90 degrees, correlated well with other phenotypic features of GRMD, such as loss of the ability to walk.

Animals↗
PubMed1993

Tethered spinal cord and an intradural lipoma associated with a meningocele in a Manx-type cat.

An 8-month-old neutered male Manx-type cat was evaluated because of clear fluid that had been draining from a skin mass dorsocaudal to the sacrum for 1 week. Neurologically, the cat had poor postural reactions and poor withdrawal reflexes in the hind limbs. Ultrasonography of the dorsal sacral area revealed a 3-cm-long hypoechoic tract extending from the skin mass cranioventrally to the area of the sacrum. The tract appeared to contain a circular mass. Results of myelography and computed tomography helped to confirm the diagnosis of a meningocutaneous tract containing a mass. Surgical exploration was performed and the tract was excised. Histologic changes were similar to those in human beings with tethered spinal cord syndrome and an intradural lumbosacral lipoma. Surgery was indicated in this cat to prevent progression of neurologic signs associated with tethered cord syndrome and to prevent problems associated with loss of CSF through a fistulated meningocele.

Animals↗