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- Author or Editor: Patrick J. Venta x
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Objective—To compare 5 methods of preparation of RNA from feline urine samples for use in a feline calicivirus (FCV), p30 gene-based, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay.
Sample Population—Urine and blood samples from 6 specific-pathogen-free cats.
Procedures—Aliquots of each urine sample (unmodified, centrifuged, or mixed with whole or hemolyzed blood) were spiked with FCV and serially diluted in urine. Serial dilutions of FCV in tissue culture medium were used as positive controls. Viral RNA was prepared via dilution and thermal inactivation (DT method), polyethylene glycol precipitation (PEG method), isolation with oligo(dT)25-coated magnetic beads (dTMB method), or extraction by use of 2 silica gel–based columns (RN or QA method). Lower detection limits and mean RT-PCR threshold cycle (Ct) values associated with each RNA preparation method and sample type were compared.
Results—Because DT-prepared samples yielded negative results via RT-PCR assay, this method was not evaluated. Lower detection limits (TCID50/sample) for the assay in urine were 1,950, 104, 11, and 7 for PEG-, dTMB-, RN-, and QA-prepared samples, respectively. For RN and QA preparations, Ct values were similar and significantly lower than those for dTMB and PEG preparations. Overall, urine modifications did not affect FCV RNA detection in dTMB-, QA-, and RN-prepared samples.
Conclusions and Clinical Relevance—Of the methods evaluated, the RN and QA methods of RNA preparation were most appropriate for the FCV RTPCR assay. An RT-PCR assay optimized for detection of FCV in feline urine may aid investigations of FCVinduced urinary tract diseases in cats. (Am J Vet Res 2005;66:915–920)
Objective—To characterize potential mechanisms of action of glucosamine inhibition of matrix metalloproteinase (MMP) expression and activity in lipopolysaccharide (LPS)-stimulated equine chondrocytes.
Sample Population—Chondrocytes cultured from samples of metacarpophalangeal articular cartilage collected from cadaveric limbs of horses.
Procedure—The effect of glucosamine on MMP activity in conditioned medium from LPS-stimulated cartilage explants was determined by a colorimetric assay with azocoll substrate. Treatments consisted of negative and positive controls, glucose (50mM), and glucosamine (50, 25, 6.25, 3, and 1.5mM). The influence of glucosamine on MMP synthesis was determined in chondrocytes in pellet culture incubated with LPS (20 µg/mL). Concentration of MMP-13 was quantified in spent medium via ELISA; nonspecific MMP activity was determined via azocoll digestion in organomercurial- activated medium. Effects of glucosamine on MMP mRNA concentration in similarly treated chondrocytes were determined by northern blot hybridization with MMP-1, -3, and -13 probes. Statistical analyses were performed with 2-way ANOVA.
Results—Glucosamine had no effect on activated MMP activity but inhibited MMP protein expression, as determined by azocoll digestion (glucosamine, 3 to 50mM) and MMP-13 ELISA (glucosamine, 1.5 to 50mM). Resting mRNA concentrations for MMP-1, -3, and -13 mRNA were significantly lower in cultures exposed to glucosamine at concentrations of 50 and 25mM than those of positive controls.
Conclusions and Clinical Relevance—Glucosamine appears capable of pretranslational, and possibly also translational, regulation of MMP expression; data suggest a potential mechanism of action for chondroprotective effects of this aminomonosaccharide. ( Am J Vet Res 2003;64:666–671)
Objective—To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1β-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro.
Sample Population—Cultured equine chondrocytes.
Procedure—Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1β (reIL-1β) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subsequent experiments, gene expression was similarly quantified from mRNA isolated from cultures pretreated with phenylbutazone to quench endogenous PGE2 synthesis, followed by exposure to reIL-1β and exogenous PGE2 (5 mg/ml) with appropriate controls.
Results—Exogenous PGE2 (10 mg/ml) significantly reduced reIL-1β-induced expression of MMP 1, MMP 3, MMP 13, and TIMP 1. Abrogation of cytokine induction with this dose of PGE2 was comparable to that for dexamethasone (10–5 M) control. Similarly, pretreatment with phenylbutazone, followed by exposure to reIL-1β and PGE2 (5 mg/ml), was associated with a reduced expression of the genes of interest, an effect that was significant for MMP 1, MMP 13, and TIMP 1.
Conclusions and Clinical Relevance—The MMP and TIMP 1 are important mediators in the pathophysiologic events in osteoarthritis. The potential for physiologically relevant regulation of expression of these genes by PGE2 is a consideration in the use of drugs that inhibit prostanoid synthesis in the treatment of equine arthropathies. (Am J Vet Res 2002;63:987–993)
Objective—To determine the effects of recombinant equine interleukin -1β (reIL-1β) and 4 anti-inflammatory compounds on the expression and activity of cyclooxygenase (COX)-2 in cultured equine chondrocytes.
Sample Population—Articular cartilage from 9 young adult horses.
Procedure—Reverse transcriptase-polymerase chain reaction methods were used to amplify a portion of equine COX-2 to prepare a cDNA probe. Northern blot analysis was used to quantify the expression of COX-2 in first-passage cultures of equine articular chondrocytes propagated in media containing dexamethasone (DEX), phenylbutazone (PBZ), polysulfated glycosaminoglycan, and hyaluronan, each at concentrations of 10 and 100 µg/ml and each with or without reIL-1β. A commercial immunoassay was used to determine prostaglandin E2 (PGE2) concentrations in conditioned medium of similarly treated cells to quantify COX-2 activity.
Results—Addition of reIL-1β increased the expression of COX-2 in a dose-dependent manner, which was paralleled by an increased concentration of PGE2 in culture medium. Concentration of PGE2 in spent medium from reIL-1β-treated chondrocytes was significantly reduced by DEX and PBZ; however, only DEX significantly reduced gene expression of COX-2.
Conclusions and Clinical Relevance—Prostaglandin E2 is considered to be an important mediator in the pathophysiologic processes of arthritis, and cultured chondrocytes respond to interleukin-1 with enhanced expression and activity of COX-2. Palliative relief in affected horses is probably attributable, in part, to inhibition of PGE2 synthesis; however, analysis of these data suggests that of the 4 compounds tested, only DEX affects pretranslational regulation of the COX-2 gene in cultured equine chondrocytes. (Am J Vet Res 2002;63:1134–1139)