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  • Author or Editor: Jeffrey S. Bartlett x
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Abstract

Objective—To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow–derived mesenchymal stem cells (BMD-MSCs).

Sample Population—Articular cartilage, synovium, and bone marrow from 15 adult horses.

Procedures—Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1–deficient adenovirus vector encoding the β-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1–deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection.

Results—Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors.

Conclusion and Clinical Relevance—Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.

Full access
in American Journal of Veterinary Research

Summary

Veterinary diagnostic endocrinology laboratories frequently receive hemolyzed plasma, serum, or blood samples for hormone analyses. However, except for the previously reported harm done by hemolysis to canine insulin, effects of hemolysis on quantification of other clinically important hormones are unknown. Therefore, these studies were designed to evaluate effects of hemolysis on radioimmunoassay of thyroxine, 3,5,3’-triiodothyronine, progesterone, testosterone, estradiol, cortisol, and insulin in equine, bovine, and canine plasma. In the first experiment, hormones were measured in plasma obtained from hemolyzed blood that had been stored for 18 hours. Blood samples were drawn from pregnant cows, male and diestrous female dogs, and male and pregnant female horses. Each sample was divided into 2 equal portions. One portion was ejected 4 times with a syringe through a 20-gauge (dogs, horses) or 22-gauge (cows) hypodermic needle to induce variable degrees of hemolysis. Two subsamples of the blood were taken before the first and after the first, second, and fourth ejections. One sub sample of each pair was stored at 2 to 4 C and the other was stored at 20 to 22 C for 18 to 22 hours before plasma was recovered and stored at —20 C. The second portion of blood from each animal was centrifuged after collection; plasma was recovered and treated similarly as was blood. Concentrations of thyroxine in equine plasma, of 3,5,3’-triiodothyronine, estradiol, and testosterone in equine and canine plasma, and of cortisol in equine plasma were not affected by hemolysis. Storage of bovine blood at either temperature and equine blood at 20 to 22 C caused progesterone concentrations to decrease (P < 0.05); the effect was not enhanced or diminished by hemolysis. Insulin concentration in equine blood decreased (P < 0.05) at both temperatures; this effect was exacerbated by hemolysis. In the second experiment, blood samples from horses and dogs were hemolyzed and plasma was immediately recovered and stored for 18 to 22 hours at 2 to 4 C or 20 to 22 C. Storage of hemolyzed equine plasma did not affect concentrations of progesterone, insulin, or thyroxine at either temperature. Whereas progesterone concentration was not affected in hemolyzed canine plasma, hemolysis decreased (P < 0.05) insulin concentration when plasma was stored at 20 to 22 C. These results emphasize the importance of examining effects of sample collection and handling procedures on hormone stability and the danger of extrapolating results of such studies from one species to another and from one hormone to another.

Free access
in American Journal of Veterinary Research