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  • Author or Editor: Mark E. Haskins x
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Abstract

Objective

To develop a molecular diagnostic test to ascertain genotype of the mucopolysaccharidosis type VII (MPS VII) locus.

Sample Population

Blood samples from 45 mixed-breed (German Shepherd Dog X Beagle) dogs that were phenotypically normal or affected with MPS VII.

Procedure

The canine ß-glucuronidase gene (exon 3) was amplified by polymerase chain reaction (PCR), using 2 pairs of primers to determine the genotype of the MPS VII locus by 2 independent methods. For the first method, PCR products were used for heteroduplex analysis, using conformation-sensitive gel electrophoresis. In the second method, an allele-specific restriction site was created by use of a mismatch primer in PCR. The amplified DNA fragment was digested with a restriction enzyme (Eag I) to enable identification of the wild-type and mutant alleles.

Results

Conformation-sensitive gel electrophoresis resulted in a single DNA band representing homoduplex when the sample contained a wild-type or MPS VII allele, but 2 bands representing hetero- and homoduplexes when both alleles were in the sample. Restriction digestion of the DNA fragment obtained by use of a mismatch primer was cleaved only when the template was a wild-type allele. Thus, samples from phenotypically normal carrier dogs that contained wild-type and MPS VII alleles were partially digested by the enzyme.

Conclusions

The diagnostic test used 2 strategies for independently ascertaining the wild-type or mutant MPS VII alleles in dogs. Thus, test results can distinguish phenotypically normal MPS Vll-carrier dogs from homozygous normal dogs. (Am J Vet Res 1998;59:1092-1095)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To determine matrix metalloproteinase (MMP) activity in synovial fluid (SF) obtained from the joints of dogs with degenerative joint disease (DJD) secondary to various underlying conditions.

Sample Population—35 samples of SF obtained from 18 clinically normal (control) dogs and 34 samples of SF obtained from 17 dogs with DJD; dogs with DJD were from 2 populations (client-owned dogs and research dogs that had DJD secondary to the lysosomal storage disease mucopolysaccharidosis VII).

Procedure—MMP activity in samples of SF was semiquantitatively examined by use of gelatin or casein zymography. Western blot analysis was performed by use of antibodies for MMP-2 and MMP-9. In addition, in situ MMP activity was observed in sections of synovial membrane obtained from healthy and osteoarthritic joints.

Results—Samples of SF from osteoarthritic joints had higher MMP-2 activity and dramatically increased MMP-9 activity, compared with values for healthy joints. Substrate-overlay analyses indicated minimal gelatin-degrading activity in synoviocytes obtained from control dogs, whereas greater activity was seen in osteoarthritic synoviocytes, with additional activity in the underlying tissue.

Conclusions and Clinical Relevance—Higher MMP-2 activity and dramatic increases in MMP-9 activity were associated with the osteoarthritic state, even though MMP-2 activity was detected in healthy joints. This study expands information on MMP production in SF of osteoarthritic joints in other species and documents the similarity of MMP activity patterns regardless of the cause of DJD. (Am J Vet Res 2003;64:1225–1233)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To examine age-related efficacy of bone morphogenetic protein (BMP)-2, ascorbate, and dexamethasone as osteogenic inducers in canine marrow-derived stromal cells (MSCs).

Sample Population—Samples of femoral bone marrow obtained from 15 skeletally immature (< 1 year old) and 4 skeletally mature (> 1.5 years old) dogs.

Procedure—First-passage canine MSC cultures were treated with 100 µg of ascorbate phosphate/mL, 10–7M dexamethasone, 100 ng of BMP-2/mL, or a combination of these osteoinducers. On day 6, cultures were harvested for quantitation of alkaline phosphatase (ALP) activity and isolation of RNA to prepare cDNA for real-time polymerase chain reaction analyses of osteoblast markers.

Results—Early markers of osteogenesis were induced in canine MSCs by BMP-2 but not dexamethasone. In young dogs, the combination of BMP- 2 and ascorbate yielded the highest ALP mRNA concentrations and activity. This combination also induced significant increases in mRNA for osteopontin and runt-domain transcription factor 2. In comparison to MSCs from immature dogs, those from mature dogs had diminished ALP activity in response to BMP and ascorbate. Results for cultures treated with 3,4-dehydroproline suggested that ascorbateinduced production of extracellular matrix was important for maximal BMP-2 response in canine MSCs.

Conclusions and Clinical Relevance—BMP-2 was capable of inducing markers of osteogenesis in shortterm cultures of canine MSCs. In MSCs obtained from skeletally immature dogs, ascorbate was required for maximal effects of BMP. These results define optimal conditions for stem cell osteogenesis in dogs and will facilitate development of stem cell–based treatments for dogs with fractures. (Am J Vet Res 2005;66:1729–1737)

Full access
in American Journal of Veterinary Research