Equine Synovial Fluid Research Articles

Automated counting of nucleated cells in equine synovial fluid without and with hyaluronidase pretreatment

Microscopy is usually used to obtain manual total and differential cell counts in equine synovial fluid. A faster, more precise method is desirable. The objectives were to compare an automated impedance method with a manual method for obtaining total and differential cell counts in equine synovial fluid and to evaluate the effect of pretreatment with hyaluronidase on automated results. Synovial fluid samples (n=48) were collected into EDTA and analyzed within 48 hours. Automated total and differential cell counts were evaluated using a Medonic CA620-VET hematology analyzer before and after pretreatment for 5-30 minutes with hyaluronidase (final concentration 0.01 mg/mL). A hemacytometer count and microscopic evaluation of a direct smear were used as the reference method. Intra-assay coefficients of variation (CV) were determined. Thirty-one of 46 untreated samples and 0/46 hyaluronidase-treated samples were error-flagged by the analyzer. Correlation between automated (ANCC) and manual (MNCC) nucleated cell counts in untreated samples (n=15; R(2)=0.93) and pretreated samples (n=46; R(2)=0.94) was high, and pseudomedian difference was low. Intra-assay CVs for samples with medium and high cellularity were significantly lower for ANCC (1.5-2.7%) compared with MNCC (6.1-15.7%) (P<.01). Valid automated differential cell counts were not obtained. Automated total cell counts obtained on the Medonic analyzer correlate well with manual counts in equine synovial fluid; however, pretreatment with hyaluronidase is required to minimize error flags. Automated differential counts are not accurate for synovial fluid.

Cartilage shear dynamics during tibio-femoral articulation: effect of acute joint injury and tribosupplementation on synovial fluid lubrication

To determine the effects of acute injury and tribosupplementation by hyaluronan (HA) on synovial fluid (SF) modulation of cartilage shear during tibio-femoral articulation. Human osteochondral blocks from the lateral femoral condyle (LFC) and tibial plateau (LTP) were apposed, compressed 13%, and subjected to sliding under video microscopy. Tests were conducted with equine SF from normal joints (NL-SF), SF from acutely injured joints (AI-SF), and AI-SF to which HA was added (AI-SF+HA). Local and overall shear strain (E(xz)) and the lateral displacement (Deltax) at which E(xz) reached 50% of peak values (Deltax(1/2)) were determined. During articulation, LFC and LTP cartilage E(xz) increased with Deltax and peaked when surfaces slid, with peak E(xz) being maintained during sliding. With AI-SF as lubricant, surface and overall Deltax(1/2) were approximately 40% and approximately 20% higher, respectively, than values with NL-SF and AI-SF+HA as lubricant. Also, peak E(xz) was markedly higher with AI-SF as lubricant than with NL-SF as lubricant, both near the surface (approximately 80%) and overall (50-200%). Following HA supplementation to AI-SF, E(xz) was reduced from values with AI-SF alone by 30-50% near the surface and 20-30% overall. Magnitudes of surface and overall E(xz) were markedly (approximately 50 to 80%) higher in LTP cartilage than LFC cartilage for all lubricants. Acute injury impairs SF function, elevating cartilage E(xz) markedly during tibio-femoral articulation; such elevated E(xz) may contribute to post-injury associated cartilage degeneration. Since HA partially restores the function of AI-SF, as indicated by E(xz), tribosupplements may be beneficial in modulating normal cartilage homeostasis.

Effect of hyaluronic acid amide derivative on equine synovial fluid viscoelasticity

An amphiphilic hyaluronic acid (HA) derivative has been obtained by the amidation of the carboxylic group of the glucuronic acid. This derivative, HYADD4-G (HY4), is the hexadecylamide of 500-730 kDa hyaluronic acid, derived from Streptococcus equi at about 2% degree of substitution (2 mol hexadecylamine per 100 mol hexuronic acid). Its viscoelastic properties, at a concentration of 5 mg/mL in phosphate buffer saline, have been compared with those solutions of native HA, having the same molecular weight. Changes in the viscoelastic properties of equine synovial fluid (SF) when mixed with HY4 over a series of volume ratios-viz 1:2, 1:1, 3:1, and 7:1-have been evaluated. HY4 is able to associate into aqueous solution, and its rheological behavior is typical of a weak gel. Throughout the frequency range investigated (0.1-10 Hz), the elastic modulus G' is higher than the viscous modulus G'', and both moduli are frequency independent, and G' value is about two orders of magnitude higher than that of a comparable solution of native HA. The addition of HY4 to equine synovial fluid (SF) increased its viscoelasticity at all the SF:HY4 ratios tested. These results demonstrate that HY4 is able to integrate with SF, increasing the synovial fluid rheology, and could be an interesting new option in viscosupplement therapy of osteoarthritis, particularly considering its low degree of chemical modification from native HA.

Evaluation of an automated blood culture system for the isolation of bacteria from equine synovial fluid

The objective of this study was to evaluate an automated blood culture system for the isolation of microorganisms from infected equine synovial fluid (SF). Samples were collected from 220 severely inflamed synovial joints and classified as either presumably infected (group A: n=149) or not infected (group B: n=71), based on a combination of clinical history, clinical signs and cytological analysis of the SF. Samples were inoculated into blood culture bottles and after incubation were subcultured onto agar media to confirm the results and to facilitate full bacterial identification. Microorganisms were isolated from 107 group A samples (71.8%) and from three group B samples (4.2%). Overall, the detection system identified 117 bottles as positive and 103 as negative, including nine instrument-false-positives and two instrument-false-negatives. The median time-to-detection for Gram-positive bacteria, Gram-negative bacteria, and for fungi was 14.3 (interquartile range [I.R.] 10.0) h, 8.8 (I.R. 12.8) h, and 72.0 (range 60.8-74.8) h, respectively. It was concluded that culture of infected SF using the automated system combines the advantages of enrichment in specialised medium with the rapid detection of bacterial growth.

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum

Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n=100) and sera (n=100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n=95) and horses (n=50). Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K63 and K238 of the EGF-like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 x biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.

Cartilage‐derived retinoic acid‐sensitive protein in equine synovial fluid from healthy and diseased joints

More sensitive and specific diagnostic methods for early detection of changes in the joint cartilage are needed. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a potential marker of cartilage synthesis and regeneration. This is the first study on equine CD-RAP. To evaluate the ability of a commercially available human sandwich ELISA assay to detect equine CD-RAP in synovial fluid from healthy and diseased joints. Synovial fluid was collected from 28 horses with no signs of joint disease and from 5 with induced inflammatory arthritis. CD-RAP concentrations were measured using a human CD-RAP ELISA. Intra- and interassay imprecision of the assay were evaluated by multiple measurements on pools of equine synovial fluid. Assay inaccuracy was determined by linearity under dilution. The assay showed moderate to large intra- and interassay variation when applied to equine synovial fluid. Equine CD-RAP was detected in synovial fluid from healthy horses ranged at 8.2-52 ng/ml. Repeated arthrocentesis (after injection of isotonic saline), age, joint or gender did not significantly affect CD-RAP concentrations. Twelve hours after intra-articular injection of lipopolysaccharide, concentrations of CD-RAP were significantly lower than after injection of isotonic saline and remained significantly lower until the end of the study at 144 h. The assay is suitable for longitudinal monitoring of CD-RAP concentration in individual horses. Disease significantly influenced CD-RAP levels. Similar to previous results obtained in man, CD-RAP seems to be a marker of cartilage synthesis and/or regeneration in horses.

1H NMR investigation of normal and osteoarthritic synovial fluid in the horse

Proton magnetic resonance spectroscopy (1H NMR) has been successfully used in the study of many biological fluids. The data presented here report on the metabolic profiles of normal equine synovial fluids compared with osteoarthritic (OA) fluids. Twenty-five OA synovial fluid samples and eight normal ones were collected from the forelimb fetlock joint in 22 horses, aged between five and 24 years. 1H NMR spectroscopy was carried out with a Bruker Avance DRX 500 equiped with a cryo-magnet working at 11 Tesla, and 'Mestre-C 4.9.9.6' software was used to analyze the spectra. The study assessed the increase of lactate, alanine, acetate, N-acetylglucosamine, pyruvate, citrate, creatine/creatinine, glycerol, HDL choline, and a-glucose in OA synovial fluid. The variations observed in samples from horses with OA compared to those in the control group, and similar data found in other studies, confirm that this technique may be useful in the study of joint metabolism. Its practical application may be in the evaluation of the treatment of OA in athletic horses.

Joint dependent concentrations of bone alkaline phosphatase in serum and synovial fluids of horses with osteochondral injury: an analytical and clinical validation

Validate use of a commercially available immunoassay for measurement of bone alkaline phosphatase (BAP) in equine serum and synovial fluid (SF), and investigate the effects of osteochondral (OC) injury in horses on BAP concentrations in serum and SF. SF was collected from 37 joints of 34 Thoroughbred (TB) racehorses undergoing arthroscopic surgery for the removal of OC fragments from either the carpal joints (n=18) or the metacarpo-/metatarsophalangeal (MP) joints (n=19). SF was also obtained from 52 joints of 16 normal TB horses, collected bilaterally from carpal joints of 10 horses (n=40), and MP joints of six horses (n=12). Blood was obtained from all 50 horses. A commercially available immunoassay was validated and subsequently used to determine equine serum and SF BAP concentrations. Correlations to radiographic and arthroscopic scores were assessed. BAP concentrations were significantly lower in serum from horses with OC injury in their carpal or MP joints than in serum from normal horses. SF BAP concentrations in normal and OC injured carpal joints were significantly higher than MP joints. BAP concentrations were significantly higher in SF from OC injured carpal joints than normal. BAP concentrations were affected by joint sampled, with age having a significant interaction. Concentrations of BAP in the serum (<30U/L), SF (>22U/L) and a ratio of SF to serum > or = 0.5 were predictive of OC injury. Radiographic and arthroscopic scores significantly correlated with serum BAP concentrations, and SF:serum BAP correlated with arthroscopic scores. Determination of serum and SF BAP concentrations may be beneficial in the investigation of early joint injury. Joint and injury dependent differences in BAP concentrations allowed the estimation of predictive value for identifying OC injury.

Molecular characterization and chromosomal assignment of equine cartilage derived retinoic acid sensitive protein (CD–RAP)/melanoma inhibitory activity (MIA)

Cartilage-derived retinoic acid sensitive protein (CD–RAP) also known as melanoma inhibitory activity (MIA) has already been established as a marker for chondrocyte differentiation and a number of cancerous conditions in humans. Studies have also shown that CD–RAP/MIA is a potential marker of joint disease. The objective of this study was to characterize the equine CD–RAP/MIA gene and thus make it available as a marker in cartilage research and clinical studies. Gene analysis revealed that the equine gene (GenBank accession no. EF679787) consists of four exons and three introns, and the homology to the human gene is 90% for the translated region. The upstream sequence includes regulatory elements and putative transcription factor binding sites previously described in the human and murine promoter regions. The deduced amino acid sequence consists of 130 aa including a signal peptide of 23 aa, and has a 91% identity to the human protein. Using radiation hybrid mapping, the CD–RAP/MIA gene was localized to the p arm of equine chromosome 10 (ECA10p), which is in accordance with prediction based on the current human-equine comparative map. Gene expression studies showed expression of CD–RAP/MIA mRNA in articular cartilage and chondrocytes from horses with no signs of joint disease. The expression decreased as the cells dedifferentiated in monolayer culture. We also identified an equine CD–RAP/MIA splice variant similar to that reported in humans. The CD–RAP/MIA protein was detected in equine synovial fluid, serum and culture medium from chondrocyte cultures. In conclusion, CD–RAP/MIA is expressed in equine cartilage and chondrocytes, and the protein can be detected in equine serum, synovial fluid and in culture medium from chondrocyte cultures. The equine gene and resulting protein share great homology with the human gene, making future studies on CD–RAP/MIAs potential as a marker in joint disease possible using the equine joint as a model.

Boundary mode frictional properties of engineered cartilaginous tissues

Despite the fact that lubrication is a primary function of articular cartilage, there is little information on the frictional properties of cartilaginous engineered tissues. A biochemical mediator of cartilage frictional properties in boundary lubrication, lubricin, has been shown to be secreted from chondrocyte-hydrogel constructs. In the current studies we utilized articular chondrocytes (CON), meniscal fibrochondrocytes (MEN), and mesenchymal stem cells (MSC) in alginate cultures to determine lubricin localization and the inherent boundary lubrication friction coefficient. Additionally, we investigated the ability of these tissues to be lubricated by synovial fluid and the reversibility of this lubrication. Cell-alginate constructs were cultured over six weeks, culture medium assayed for lubricin release by ELISA and constructs analyzed with immunohistochemical (IHC) methods to investigate the localization of lubricin. Engineered tissues were tested in a custom friction instrument to determine the equilibrium friction coefficient (microeq) in boundary lubrication mode, following incubation with equine synovial fluid (SF), and subsequent extraction in l.5M NaCl. MSCs released 10 fold more lubricin than CON or MEN cultures. IHC analysis showed no localization of lubricin to alginate, minimal focal staining of engineered constructs at six weeks in culture, and the ability of all engineered tissues to localize lubricin when exogenously treated with SF. Frictional characterization showed no difference in microeq over culture for all engineered tissues, while incubation in SF decreased microeq for all tissues over culture duration, and extraction of lubricin resulted in a loss of lubrication of all engineered tissues.

Características físico-químicas e citológicas do líquido sinovial da bainha tendínea digital de eqüinos

Foram estudadas as características físico-químicas e citológicas do líquido sinovial da bainha tendínea digital de nove eqüinos hígidos. Verificou-se que o líquido é viscoso, amarelo claro, límpido, livre de partículas e que não coagula à temperatura ambiente. Sua concentração média de ácido hialurônico foi 60,20mg/dl, a taxa de glicose, similar à plasmática e sua concentração protéica não ultrapassou 1,74g/dl, com relação média albumina:globulina de 0,94. O número médio de células nucleadas foi de 313 células/µl, com predominância de grandes células mononucleares e linfócitos. Houve correlação significativa (r = - 0,649, P&lt;0,01) entre o aumento da concentração de ácido hialurônico e a diminuição percentual de linfócitos. As mensurações das características pesquisadas no líquido sinovial da bainha tendínea digital de eqüinos são de execução simples e passíveis de implantação na rotina de atendimentos clínico-cirúrgicos.

Evaluation of hyaluronidase activity in equine and bovine sera and equine synovial fluid samples by use of enzyme zymography

To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes. Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses. Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis. [corrected] Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7 Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes. Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints.

Collagenase‐1 (MMP‐1) activity in equine synovial fluid: influence of age, joint pathology, exercise and repeated arthrocentesis

Matrix metalloproteinases (MMPs) are considered candidate biomarkers for both physiological and pathological tissue remodelling because of their key role in articular cartilage homeostasis. As disruption of the collagenous architecture is thought to be pivotal in chronic degenerative diseases such as osteoarthritis (OA), the collagenases form an interesting subset of the MMPs. The significance of any biomarker in synovial fluid (SF) can be assessed properly only when fluctuations in patterns induced by physiological processes such as development and growth, and by external influences and interventions such as exercise and repeated arthrocentesis, are known and taken into account. To investigate the activity of MMP-1 in equine SF at different stages of development and in joints affected by OA, and the influence of exercise and repeated arthrocentesis thereon. MMP-1 activity was determined in SF of normal joints of fetal, juvenile and mature horses, and in SF of horses suffering from OA, using an internally quenched fluorogenic peptide substrate. MMP-1 activity was also measured in SF from horses subjected to an exercise regimen and those subjected to repeated arthrocentesis. An age-related decline in the SF levels of active MMP-1 was observed. MMP-1 activity was 15-fold higher in fetal than in juvenile animals, which showed significantly higher MMP-1 activity levels than mature horses. In SF of OA joints, MMP-1 activity was increased. Exercise did not affect MMP-1 activity in SF, but repeated arthrocentesis (within 60 h) increased MMP-1 activity significantly. The high MMP-1 activity in SF of young individuals parallels the high metabolic activity occurring during rapid growth and differentiation at early age. The elevated MMP-1 activity in SF of OA joints probably reflects pathological matrix degradation, confirming the potential of MMP-1 to serve as a biochemical marker for early joint disease. Moderate exercise is not likely to influence the outcome of MMP-1 activity measurements in equine SF, but arthrocentesis should be taken into account as a possible confounding factor. Given the crucial role of the collagen matrix for tissue integrity, MMP-1 activity may be a useful tool in diagnostic, therapeutic or prognostic studies in horses suspected of OA. However, care should be taken to exclude fluctuations in MMP-1 activity induced by physiological processes such as development and growth, and by interventions such as repeated arthrocentesis.

Is SAPL the boundary lubricant in prosthetic joints: friction testing and surface rinsing

The purpose of this study was to test whether surface-active phospholipid (SAPL) has the potential to decrease friction in the prosthetic joint by acting as a boundary lubricant. The specimens used were ultra high molecular weight polyethylene (UHMWPE) pins and highly polished (surface roughness of 0.01 to 0.02 μm) stainless steel plates (materials commonly used in prosthetic joints). A Hounsfield test rig was used to perform two sets of experiments. The first of these experiments used equine synovial fluid as the lubricant. It was found that the coefficient of friction (μ) was quite high, between 0.12 and 0.18. The second experiment was comparative testing using synthetic dipalmitoyl phosphatidylcholine (DPPC), calcium chloride (CaCl2), saline, and hyaluronic acid (HA) as the lubricants. It was found that when DPPC was used the friction was halved (μ changed from 0.12 to 0.05), suggesting that DPPC acts as an effective boundary lubricant. Reciprocating tests using a purpose built apparatus were also performed using DPPC and saline in order to assess the friction over an extended period of time. It was observed that the friction obtained was comparable to the Hounsfield friction tests using DPPC (coefficient of friction in the range of 0.05 to 0.07). Surface rinsings were performed on the specimen after soaking in equine synovial fluid to determine what had absorbed to the surfaces and the quantity that had absorbed. Only small amounts of DPPC and sphingomyelin, also a phospholipid, were found, due to the lack of SAPL in the synovial fluid, suggesting why such high values of friction prevailed in the first Hounsfield experiment. The above experiments strongly suggest that SAPL has the ability to decrease friction within the prosthetic joint by acting as a boundary lubricant.

Development of a solid-phase assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid.

To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays. 25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses. A solid-phase DMMB assay was developed to measure sGAG concentrations in SE Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Famdale method) and microplate DMMB assay. The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solid-phase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays. The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solid-phase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses.

Synovial fluid cytokines and eicosanoids as markers of joint disease in horses.

To evaluate the value of various synovial fluid cytokines and eicosanoids to diagnose joint disease or categories of joint disease. Prospective acquisition of clinicopathologic data. Client-owned or donated horses: 50 joints with no evidence of disease; 28 joints with acute disease; 32 joints with chronic disease; 9 joints with cartilage damage and no other signs of joint disease. Concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), prostaglandin F1-alpha (PGF(1)-alpha), and leukotriene B(4) (LTB(4)), were measured in equine synovial fluid by immunoassay and categorized according to duration and degree of joint disease. Any test value for a given category that was different from normal was further analyzed for sensitivity (S), specificity (Sp), and operating point (most valid test cutoff value). Likelihood ratios and predictive values were calculated at the operating point. Mediator concentrations were correlated to synovial fluid white blood cell count. Tests were reported as poor, fair, good, or excellent based on predictive values of <.25,.25-.5,.5-.75, or >.75, respectively. TNF synovial fluid concentration as a predictor of joint disease was good, and the value of TNF (maximum S and Sp) indicating joint disease was >36 pg/mL. IL-1beta as a predictor of joint disease was good, and the value of IL-1beta indicating joint disease was >4.5 pg/mL. IL-6 concentration was an excellent predictor of joint disease. Any IL-6 in synovial fluid indicated joint disease and correlated highly with synovial fluid white blood cell count (P <.0001). PGE(2) was a good-excellent predictor of disease (positive predictive value [PPV] = 0.75), and the concentration indicating joint disease was >22.5 pg/mL. The diagnostic PGF(1)-alpha concentration indicating severe chronic joint disease was identified to be >16.5 pg/mL with very high sensitivity (S = 1) and specificity (Sp =.89). PGF(1)-alpha concentrations > 9.5 pg/mL had a good PPV (.69) and NPV (.6) for any joint disease. TBX(2) concentrations below 31.5 pg/mL (S =.57; Sp =.61) were a very good predictor of joint disease (PPV =.72). LTB(4) concentration appeared to be greater in severe acute joint disease than normal joints; this was not significant (P =.15) and correlated highly with synovial fluid white blood cell count (P =.0001). The ability of a single value from a joint in an adult horse predicting the presence of joint disease was often good (.5-.75), and was excellent (> or =.75) for IL-6 and PGE(2). TNF-alpha and IL-1beta were no more effective than white blood cell count in screening for joint disease. IL-6 was the most sensitive and specific for joint disease and could be an excellent screening test for the presence of joint disease when lameness is difficult to identify or is intermittent. PGE(2) would be a functional screening test for the presence of any joint disease and offers a differentiating feature because values were not influenced by white blood cell count. PGF(1)-alpha values > 16.5 pg/mL identified chronic severe joint disease and may be clinically useful when there are minimal radiographic changes but substantial articular cartilage degradation.

Measurement of cartilage oligomeric matrix protein (COMP) in normal and diseased equine synovial fluids

Objective This study was designed to assay cartilage oligomeric matrix protein (COMP) in equine synovial fluids and to compare the concentration in synovial fluids from normal horses with joint diseased horses. The relationship between the COMP degradation and the matrix metalloproteinase activity in synovial fluids was also investigated.Design Using COMP antigen prepared from equine articular cartilage and murine monoclonal antibody (12C4) raised against human COMP, an inhibition ELISA was developed. COMP in equine synovial fluids from normal and diseased joints was quantified. Metalloproteinase activities were evaluated in the same synovial fluids by a gelatin degradation ELISA. COMP fragments were evaluated qualitatively by Western blotting.Results The COMP inhibition ELISA was reliable at concentrations of equine COMP between 62.5 and 2000ng/ml. COMP values in joint fluids in both aseptic and septic joint disease (19.7±15.3 and 16.1±11.2μg/ml, respectively) were significantly (P< 0.001) lower than normal (53.2±29.0μg/ml). The molecular sizes of COMP on immunoblots were different between normal and diseased synovial fluids; more fragments were seen in diseased fluids. The aseptic (26.6±20.6%) and septic joint disease synovial fluids (36.1±37.5%) had significantly higher (P< 0.02 and 0.002, respectively) gelatinolytic activities than normal (13.6±13.7%). There was a negative correlation (R=−0.31, P< 0.002) between COMP level and gelatinase activity.Conclusions We conclude that the fragment pattern and the absolute COMP concentration maybe useful for monitoring joint disease, and that COMP degradation in synovial fluids from progressed joint disease may be due to MMP gelatinolytic activity.

Equine TIMP-1 and TIMP-2: identification, activity and cellular sources.

Matrix metalloproteinases (MMPs) are the main enzymes involved in connective tissue turnover. Regulation of MMPs is achieved by controlling production, activation of the pro-enzymes together with the presence of inhibitors, such as, tissue inhibitors of metalloproteinases (TIMPS). The presence of TIMPs in equine synovial fluid was assessed by the ability of the fluid to inhibit equine MMP-9 activity using a gelatin degradation ELISA. The cellular source of the TIMPs was determined using culture supernatants of resident articular cells (chondrocytes and synovial fibroblasts) and invading inflammatory cells (polymorph neutrophils [PMN] and peripheral blood monocytes [PBM]). The TIMPs were characterised further using reverse zymography, affinity chromatography and N-terminal amino acid sequencing. Synovial fluid was recovered from horses with articular sepsis and aseptic joint disease (AJD) and compared with that from normal horses (n = 4). TIMP activity was minimal in articular sepsis but significantly increased, albeit a small increase, in AJD when compared to normal (P<0.05). Cell culture supernatants from synovial fibroblasts, chondrocytes and PBMs contained TIMP activity, although supernatants from PMN cell culture did not. Reverse zymography of synovial fluid recovered from normal and AJD horses showed two protein bands, 22 and 28 kDa in size, exhibiting inhibitory activity against MMP-9. Reverse zymography of culture supernatants of synovial fibroblasts and chondrocytes gave similar results whereas the culture supernatants from PMNs and PBMs showed the presence of only the 28 kDa protein. The N-terminal amino acid sequence was obtained for the 22 kDa protein and revealed a 66% homology with human TIMP-2. The identification of TIMPs in equine synovial fluids and cell culture supernatants suggest that they may have a fundamental role in the homeostasis of the normal joint and in the excess proteolysis which occurs in articular disease in the horse.

A Microtiter Plate Assay for the Determination of Uronic Acids

The amount of uronic acid residues in samples containing glycosaminoglycans or pectin is an important parameter in the quantitative and structural analysis of these complex carbohydrates. This paper describes a method to determine the content of uronic acids in biological samples, using conventional polystyrene microtiter plates and microtiter plate-reading equipment with standard interference filters (i.e., 540 or 492 nm). This assay is a modification of a commonly used procedure, viz. hydrolysis of uronic acid containing carbohydrate polymers in 80% sulfuric acid containing tetraborate ions at 80°C followed by a coloring step with anm-hydroxydiphenyl reagent at room temperature. The use of microtiter plates has several practical advantages: (i) less risk of handling hot, concentrated sulfuric acid is present; (ii) an accurate estimate of background absorbance by multiple reading of the plates is possible; and (iii) many samples can be assayed in one series without errors due to fading of the final color. The validity of the assay was checked for the quantification of hyaluronic acid in equine synovial fluid samples. We consider this the method of choice when a large number of samples must be analyzed for their content of uronic acid residues.

Transforming growth factor-β1 concentrations in equine synovial fluid

Summary A commercial assay for human TGF-β1 was validated for use on equine synovial fluid and serum. This assay was then used to determine concentrations of TGF-β1 in synovial fluid samples from 109 metacarpophalangeal joints of horses with varying age, gender, and joint condition. No difference in concentration was found between geldings and females. A difference (p=.04) was found between young horses (≤48 months, 1101 pg/ml) and old horses (≥120 months, 863 pg/ml), and between horses with visibly normal joints and those with severely diseased joints (885 versus 1002 pg/ml, p=0.01). These findings suggest concentrations of TGF-β1 are elevated in joints from younger horses and joints with gross evidence of disease.