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 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
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
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 (10–5M) 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
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
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)