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DATE DUE DATE DUE GLUCOSAMINE AND CHONDROITIN SULFATE INFLUENCE CATABOLIC RESPONSES TO RECOMBINANT EQUINE INTERLEUKIN-l IN EQUINE CHONDROCYTES By Kirsten Maree Neil A THESIS Submitted to Michigan State University In partial fulfillment of the requirements For the degree of MASTER OF SCIENCE Department of Large Animal Clinical Science 2005 ABSTRACT GLUCOSAMINE AND CHONDROITIN SULFATE INFLUENCE CATABOLIC RESPONSES To RECOMBINANT EQUINE INTERLEUKIN-l IN EQUINE CHONDROCYTES By Kirsten Maree Neil Joint disease, and in particular osteoarthritis, is a significant caUse of lameness and poor performance in horses. Currently, a number of pharmacological agents are available for the treatment of osteoarthritis, including glucosamine and chondroitin sulfate. Although a number of in vitro studies have been conducted using these compounds, concentrations used have exceeded those that are obtained by oral administration. A subsaturating dose of recombinant equine interleukin-l (500 pg/ml) was established that resulted in approximately half maximal induction of expression of mediators of osteoarthritis. The effect of glucosamine and chondroitin sulfate on gene expression of these mediators of osteoarthritis was investigated using equine chondrocytes in pellet culture stimulated with recombinant equine interleukin-1. Glucosamine at 10.0 pg/ml significantly reduced mRNA expression of matrix metalloproteinase 13, aggrecanase 1 and c-Jun-N-terminal kinase. Further, a trend for reduction in interleukin-1 induced expression of inducible notric oxide synthase and cyclo-oxygenase 2 was observed. Chondroitin sulfate had no effect at the concentrations tested. ACKNOWLEDGEMENTS The author thanks the American Association of Equine Practitioners for funding this project. I would like to thank Steve Suchyta for his assistance with problems that arose with the RT-PCR analysis, Peggy Wolfe and Robert Harlan for helping with RNA spectrophotometry, and especially Pooi-See Chan for her technical assistance. I would also like to thank the members of my committee for their guidance and support: Michael Orth, Paul Coussens, Elizabeth Carr and especially John Caron, for without his knowledge, commitment, patience and perseverance this project would not have been completed. iii TABLE OF CONTENTS LIST OF TABLES - vi LIST OF FIGURES _ ..... vii KEY TO ABBREVIATIONS - - -- . - iix INTRODUCTION- - 1 CHAPTER 1: LITERATURE REVIEW - 3 Synovial Joint: ........................................................................................................ q ....... 3 Structure - Function Relationships ......................................................................... 3 Synovial fluid and Synovium ................................................................................... 3 Articular Cartilage .................................................................................................... 4 Subchondral Bone ..................................................................................................... 9 Joint Biomechanics ....................................................................................................... 9 Osteoarthritis ............................................................................................................... 12 Degradative Enzymes and their Inhibitors ........................................................... l6 Inflammatory mediators ........................................................................................ 23 The Role of Cytokines ............................................................................................. 25 Drugs used in Osteoarthritis Treatment ................................................................... 38 Nonsteroidal anti-inflammatories .......................................................................... 38 Corticosteroids ........................................................................................................ 40 Hyaluronan .............................................................................................................. 41 Polysulfated Glycosaminoglycans .......................................................................... 42 Pentosan Polysulfate ............................................................................................... 43 Glucosamine and Chondroitin sulfate ...................................................................... 44 Structure .................................................................................................................. 44 Actions in Articular Tissues ................................................................................... 45 Effect on IL-1 signaling pathways ......................................................................... 54 Pharmacokinetics .................................................................................................... 55 Clinical Trials .......................................................................................................... 65 References .................................................................................................................... 75 CHAPTER 2: DETERMINATION OF A SUBSATURATING DOSE OF RECOMBINANT EQUINE INTERLEUKIN-lfl FOR GENES IMPLICATED IN CARTILAGE DEGRADATION IN OSTEOARTHRITIS 115 Summary .................................................................................................................... 115 Introduction ............................................................................................................... 116 Materials and Methods ............................................................................................. 118 Tissue Sources-Pellet Cultures ............................................................................... 118 RNA Isolation ......................................................................................................... 120 Quantitative Real Time PCR .................................................................................. 120 Stimulation with reIL-l B ........................................................................................ 122 iv Results ........................................................................................................................ 123 Inflammatory mediators .......................................................................................... 123 Extracellular Matrix Proteins .................................................................................. 123 Transcription Factors .............................................................................................. 123 Degradative Enzymes and their inhibitors ........... Error! Bookmark not defmed.124 Discussion .................................................................................................................. 125 CHAPTER 3: GLUCOSAMINE AND CHONDROITIN SULFATE REGULATION OF MEDIATORS OF OSTEOARTHRITIS IN RECOMBINANT EQUINE INTERLEUKIN-IB STIMULATED EQUINE CHONDROCYTES IN PELLET CULTURE. 140 Summary .................................................................................................................... 140 Introduction ............................................................................................................... 141 Materials and Methods ............................................................................................. 144 Tissue Sources- Pellet Culture ................................................................................ 144 RNA Isolation ......................................................................................................... 145 Determination of a Sub-saturating Dose of reIL-l B ............................................... 148 Selection of housekeeping genes for use as endogenous controls in subsequent studies ..................................................................................................................... 148 Effect Of Glucosamine and Chondroitin Sulfate on Gene Expression ................... 149 Statistical Analysis .................................................................................................. 149 Results ........................................................................................................................ 150 Determination of a Sub-saturating Dose of reIL-l B ............................................... 150 Selection of housekeeping genes for use as endogenous controls in subsequent studies ..................................................................................................................... 150 Effect Of Glucosamine and Chondroitin Sulfate on Gene Expression ................... 151 Discussion ................................................................................................................... 151 References .................................................................................................................. 165 CHAPTER 4: QUANTIFICATION OF GENE EXPRESSION USING A SUBSATURATING MODEL OF OSTEOARTHRITIS: CONSIDERATIONS FOR ENDOGENOUS CONTROLS AND EXPERIMENTAL DESIGN 171 References ......................................................................................... 183 LIST OF TABLES Table I (chapter 2): Forward and reverse primer sequences (S’—>3’) used for quantitative real-time polymerase chain reaction ....................................................... page 130 Table I (chapter 3): Forward and reverse primer sequences (5’—>3’) of housekeeping genes used for quantitative real-time polymerase chain reaction ...................... page 160 Table II (chapter 3): Forward and reverse primer sequences (5’—>3’) of genes of interest used for quantitative real-time polymerase chain reaction ............................. page 161 Table I (chapter 4): Forward and reverse primer sequences (5’—>3’) of genes of interest used for quantitative real-time polymerase chain reaction ............................ page 181 Table 11 (chapter 4): Suitability of housekeeping genes for each gene of interest based on amplification efficiency. The value of the slope of the regression of the plot of ACT versus log cDNA dilution was used as a measure of the difference in amplification efficiencies between the gene of interest and each housekeeping gene. A difference in efficiency of 20 cm H20), the slope of the pressure-volume 10 curve changes rapidly with decreasing compliance and greater pressure increases with small increases in volume. Normal synovial membrane is elastic at low to moderate pressures, dissipating energy during the load/unload cycle (hysteresis). Compressive stiffness of articular cartilage is related to proteoglycan content and quality. Because the load born by different areas of cartilage varies, mechanical properties and morphology vary topographically (Murray et al 1999). Highly congruent joints generally have thinner cartilage, while lower congruency centers the applied load over a smaller surface area. The effect of loading on articular cartilage depends on the duration and magnitude of the applied load. Moderate exercise stimulates proteoglycan synthesis (Palmer et al 1995a), increases GAG concentration (Brama et al 1999a), and enhances mechanical stiffness, whereas strenuous exercise induces deleterious effects. Increasing exercise intensity increases cartilage water content and reduces collagen cross-linking (Brama et al 1999c). Subchondral bone remodeling and thickening of the calcified layer of articular cartilage alter joint biomechanical properties (Muller-Gerbl et a1 1987). The response of articular cartilage to loading depends on both the location within the joint and exercise intensity level. Under load, mean pressure and contact area increase on the dorsal aspect of the radial facet of the equine third carpal bone, corresponding to the area subjected to the most trauma in racing horses (Palmer et a1 1994). Consequently, the structure and hence function of cartilage overlying the dorsal aspect of the radial facet is altered, with reduced stiffness and proteoglycan content (Palmer et al 1995). Dorsal sites in the carpus are 11 subjected to intermittent high loading, and, with high intensity exercise, collagen and proteoglycan content decrease. Collagen content is greater at dorsal compared to palmar sites, and GAG content higher at palmar Sites that are subjected to lower loads (Murray et al 2001). Both exercise intensity and cartilage location within a joint (due to different load distributions) influence composition and mechanical properties of articular cartilage, leading to an inherent predisposition for injury and disease. The density of subchondral bone also influences joint biomechanics. Subchondral bone remodeling and thickening of the calcified layer of articular cartilage alter joint biomechanical properties (Muller-Gerbl et a1 1987). In disease states, inflammation alters the synovial membrane, articular cartilage, and synovial fluid content and volume. As the severity of clinically evident joint disease increases, biomechanical properties are compromised. For instance, the range of motion in flexion decreases. With disease, stiffness of the soft tissue of the dorsal pouch of metacarpophalangeal joints increases along with IAP, with changes in OA less severe than with synovitis (Strand et al 1999). Osteoarthritis Joint disease is a well-known and expensive problem for the equine athlete that causes substantial morbidity in the form of lameness in affected animals and may seriously curtail their quality of life in addition to their athletic serviceability. Indeed, OA and the resultant lameness is the major reason that horses become unserviceable for racing and estimates for the cost of this problem to the racing industry are staggering. A number of 12 studies have reported that lameness is the most Significant cause of wastage both in racehorses (Bailey 1998, Jeffcott et a1 1992, Rossdale et a1 1983) and the general horse population (Kaneene et al 1997), with joint disease figuring prominently. Initially, four main categories of OA, also referred to as degenerative joint disease (DJ D), were described on the basis of joint characteristics and predisposing factors (McIlwraith 1982). This classification was modified to recognize three types of DA in the horse, namely: 1. Associated with synovitis and capsulitis. Common in both high motion joints (eg fetlock in racing Thoroughbreds) and low motion, high load joints (eg distal tarsal and distal interphalangeal joints). 2. DJD secondary to predisposing factors such as osteochondrosis, articular fractures, septic arthritis, subchondral bone injury and disease. 3. Incidental or nonprogressive idiopathic cartilage erosion. Grossly, articular cartilage degeneration is apparent as fibrillation, erosion and wear lines (McIlwraith 1996), with subchondral sclerosis, osteophyte production and synovitis also features of 0A. The progression of 0A is divided into three stages. The initial stage involves proteolytic breakdown of ECM, then fibrillation and erosion of the cartilage surface occur, accompanied by the release of ECM degradation products into the synovial fluid. Finally, synovial inflammation occurs subsequent to the production of further degradative enzymes and proinflammatory cytokines (Pelletier et al 2000). 13 The hallmark of 0A is the progressive permanent degeneration of articular cartilage. Normally a balance exists between the synthesis and degradation of the ECM. Biochemical and mechanical factors disrupt this balance, primarily by targeting chondrocytes with subsequent degeneration of the ECM (Goldring 2000). In early OA, chondrocytes transiently respond by increasing ECM synthesis in an attempt at repair. However, production of cytokines, inflammatory mediators and matrix degrading enzymes predominate, leading to PG loss, cleavage of type II collagen and degradation of aggrecan, events which are synonymous with CA. Articular cartilage lesions are accompanied by biochemical alterations including changes in matrix composition, reflecting aberrant behavior of chondrocytes. Normally, a certain amount of mechanical loading is required for joint lubrication and the stimulation of proteoglycan synthesis, with detrimental effects apparent when an optimal load is exceeded. Three types of chondral and osteochondral injuries have been identified based on the type of tissue damage and repair mechanisms (Buckwalter 2002): damage to the joint surface without visible mechanical disruption of the articular surface, but with possible subchondral bone damage; mechanical disruption of the articular surface limited to the articular cartilage; and involvement of articular cartilage and subchondral bone. In most instances, the joint can repair damage that does not disrupt the articular surface provided it is protected from additional injury. Disruption of articular cartilage stimulates chondrocyte activity but rarely is complete repair possible. Damage to subchondral bone stimulates both chondrocyte and bony repair; however, the biological and mechanical properties of the articular surface are rarely restored to normal. 14 Clinical signs of OA include lameness, joint pain, reduced range of motion, and variable joint effusion. In high motion joints, OA manifests as synovitis, cartilage erosion, and subchondral bone sclerosis; whereas in low motion joints, synovitis is less a feature; however, full thickness cartilage necrosis with little erosion and subchondral bone lysis occurs. Joint predisposition to 0A is apparent, with OA of the distal tarsal joints the most frequent cause of hock lameness in horses (McIlwraith 1996). Synovitis and capsulitis are common initial changes in joints. Damage to joint structures such as articular cartilage can result in the release of cytokines and MMPS that may then influence not only the cartilage itself but also the synovium. Conversely, synoviocytes secrete MMPS (Martel-Pelletier 1986), PGEz, free radicals and cytokines, with possible subsequent articular cartilage damage. Biological markers can be measured in both serum and synovial fluid to monitor joint metabolism, with CS epitopes (383, 7D4) released into synovial fluid in OA. Further, articular cartilage damage and synovial fluid levels of bone specific alkaline phosphatase and keratan sulfate epitopes are highly correlated in OA (Fuller et al 2001). Irreversible degradation of type II collagen is an early critical event in OA. Cytokines such as IL—1 suppress synthesis of collagen type II and IX, and increase collagen type I and III (Goldring et al 1988). Thus in OA, collagen synthesis changes from cartilaginous to fibrous type, resulting in altered biomechanics and morphological changes such as joint capsule fibrosis. Denaturation of type II collagen usually originates in superficial 15 layers of cartilage, with a close correlation between sites of cleavage and the presence of collagenases (Wu et al 2002). Further, alterations in type VI collagen occur in DA, with loss of pericellular type VI collagen in superficial zones and net increase in synthesis in middle zones (Hambach et al 1998). The breakdown of ECM components is controlled by the activity of a number of degradative enzymes, including MMPS and aggrecanases; however, inflammatory mediators also play an important role in OA. Degradative Enzymes and their Inhibitors Matrix metalloproteinases The matrix metalloproteinases are a group of zinc containing calcium dependent proteinases that are active at neutral pH. These enzymes are involved in normal physiological turnover of the ECM. In 0A, MMPS play a central role in degradation of the ECM, in part due to the loss of the normal balance between MMPS and their inhibitors. The concentration of MMPS in OA cartilage exceeds that in normal cartilage (Dean 1991, Okada et a1 1992), with production topographically related to lesion location (Martel-Pelletier et al 1994, Tetlow et al 2001 ). MMPS are divided into groups based on their substrate specificity, sequence similarity and structure. Currently, four groups have been implicated in ECM degradation, namely the collagenases, gelatinases, stromelysins and membrane-type MMPS (MT-MMPS). The collagenases (MMP 1, 8, 13) are unique in their ability to cleave the intact triple helix of collagen type I, II and III. The role of collagenases in maintaining ECM homeostasis is critical and is, in fact, the rate limiting step for fibrillar degradation. MMP 1 and 13 16 cleave type II collagen between glycine775-isoleucine776, producing a 3%: length TCA N terminal fragment and a TCB C terminal fragment. These fragments are then susceptible to further proteolysis by gelatinases (Mengshol et a1 2002). MMP 13 is more efficient, has a broader substrate Specificity and is also capable of cleaving collagen types IV, X and XIV as well as gelatin. MMP 8 is termed neutrophil collagenase; however, chondrocytes can also produce this MMP (Cole et a1 1995). The gelatinases (MMP 2, 9) cleave denatured collagen, aggrecan and link protein, and also activate other MMPS such as MMP 3 and 13. Stromelysins (MMP 3, 10, 11) degrade ECM components such as gelatin, aggrecan and fibronectin. The stromelysins exhibit similar substrate specificities; however, of the three, MMP 3 has a more efficient proteolytic capacity (Visse et a1 2003). MMP 3 can also degrade link protein and denatured type II collagen, and activate latent/inactive MMP l and 9 (proMMP 1, 9), with its action on proMMP I thought to be critical for generation of the full activity of MMP 1 (Suzuki et a1 1990). The fourth groups of MMPS, the membrane-type MMPS (MMP 14, 15, 16, 17, 24, 25), are transmembrane proteins capable of digesting ECM proteins such as aggrecan and fibronectin. MMP 14 also has collagenolytic activity, and the MT-MMPS are capable of activating proMMP 2. Structurally MMPS contain a number of domains, ie discrete amino acid sequence regions, that impart a particular function. These include a prodomain, catalytic domain, hinge region and hemopexin domain. The prodomain and catalytic domains are common to all MMPS. All MMPS, except MMP 11, are synthesized as latent proenzymes and 17 secreted in the inactive form (proMMPs). A N-terminal sequence targets MMPS for secretion. Secreted MMPS contain a prodomain and are enzymatically inactive. Within the prodomain is a conserved sequence referred to as a cysteine switch motif. This motif is responsible for the latency of proMMPs as a complex forms between its cysteine residue and the zinc-binding motif of the catalytic domain, such that the complex lies over the substrate—binding clefl of the catalytic domain. Activation, one of the important steps in MMP regulation (Ohuchi et al 1997), requires detachment of the cysteine residue, achieved via detachment of the prodomain. Certain MMPS (MMP 11, 14-17) also contain a proprotein processing sequence at the C terminal end of the prodomain that also detaches. In addition to the zinc-binding motif, the central catalytic domain also contains a unique conserved “Met-tum” structure that acts as a base to support the protein backbone that loops around the zinc residue. The catalytic domain is responsible for the proteolytic activity of MMPS, with zinc and calcium ions required for both stability and expression of activity. The C-tenninal hemopexin-like domain has a number of functions. This domain is required for the collagenases to cleave the triple helix of collagen (Nagase et al 1999), for cell surface activation of MMP 9, and is also the site where TIMPS bind. Binding to cell surface receptors by MMP 2 and 9 is achieved via 3 repeats of fibronectin-type II domains within the catalytic domain. ProMMPs can be activated by proteinases, such as tissue or plasma proteinases, and reactive oxygen species. In addition, serine proteases, such as plasma-derived urokinase- 18 type plasrrrinogen activator, can activate MMP 1 and 3 (Milner et a1 2001). Some of the serine proteases are active in OA joints, providing an in vivo mechanism for activation of latent MMPS (Kummer et al 1992). In OA, both synoviocytes and chondrocytes produce MMPS (Zafarullah et al 1993), in particular MMP 1, 3, 8, 9, and 13 (Cole et al 1996, Shlopov et a1 1997). Expression is usually restricted to Sites of active lesions, and results in destruction of type II collagen and proteoglycans. Elevated synovial fluid concentrations of MMP l, 2, 3 and 9 have been detected in equine OA joints (Clegg et a1 1997, Trumble et al 2001, Brama et a1 2004). Detection of increased MMP 3 levels in plasma of human patients with knee OA suggests that expression may not be localized to the affected joint (N aito et a] 1999). Control of MMP gene expression can occur at the transcriptional level. Many of the MMPS are inducible (eg MMP 1, 3, 9, 13) with enhanced expression under the influence of growth factors and cytokines such as TNF and IL-1. In contrast, MMP 2 is constitutively expressed, under minimal regulation and, as such, thought to have a lesser role in OA. MMP 1 and 3 are ubiquitously expressed, and MMP 3 is capable of activating MMP 1. Normally, MMP 13 expression is limited to sites of development; however, during OA, MMP 13 production increases (Billinghurst et al 1997, Reboul et a1 1996). MMP 13 is thought to play a pivotal role in OA due to its efficient hydrolysis of type II collagen (Mengshol et al 2002), with expression localized to Sites of collagen degradation (Mitchell et a1 1996). MMP 9, also produced by peripheral blood monocytes and neutrophils, is thought to play a secondary role as collagen degradation occurs only after 19 MMP 1 and 13 have cleaved the triple helix (Nagase et al 1999). MMP 8 is only expressed by articular chondrocytes in small quantities, suggesting a less important role in OA (Stremme et a1 2003). MMP expression profiles in cartilage appear to differ depending on the stage of OA. Temporal induction of MMP expression in human chondrocytes by IL-1 has been demonstrated in vitro (Koshy et a1 2002). Maximal induction of mRNA expression of MMP l, 3 and 13 is observed early (maximal 8-12 hours), but MMP 8 expression is delayed, with maximum expression 48 hours post stimulation. Further, MMP expression profiles differ in early compared to late OA cartilage. MMP 2 and 13 are upregulated in late stage cartilage, whereas MMP 3 is upregulated early and down regulated later (Aigner et al 2001, Bau 2002). Aggrecanases Cleavage of aggrecan is an important and early stage event in ECM degradation, preceding the degradation of collagen (Aigner et a1 2002). However, cleavage of collagen can then render proteoglycans susceptible to proteolysis. Catabolic factors such as IL-1 can induce degradation of aggrecan by both aggrecanases and MMPS (Sandy et a] 1991, Flannery et a1 1992, Lohmander et a] 1993). Aggrecanases have an integral role in both the normal turnover of aggrecan in cartilage, as well as in OA (Lark et al 1997). Aggrecanases are members of the ADAMTS family (A disintegrin-like and metalloproteinase domain with thrombospondin type I motifs) (Apte 2004). These 20 enzymes are a subgroup of the membrane bound ADAMS (a disintegrin and a metalloproteinase domain), but are not integral membrane proteins. Like MMPS, aggrecanases are also multi-domain enzymes containing a metalloprotease domain, the catalytic domain of which also contains a zinc binding motif, and a disintegrin-like domain along with a number of thrombospondin type-l domains. Aggrecanases l and 2 (ADAMTS 4 and 5 respectively) are synthesized as inactive enzymes, with activation requiring removal of a prodomain and removal of a C-terminal spacer domain, removal of the latter possibly mediated via MMPS (Gao et a1 2002). Degradation of aggrecan involves proteolysis in the interglobular domain near the N— terminus of the core protein, resulting in release of core protein and GAG into synovial fluid with subsequent loss of compressive resistance. Two cleavage Sites have been identified; at asparaginem-phenylalanine342 and glutamic acid373-alanine374 bonds. MMPS cleave at the first site and aggrecanases at the latter; however, aggrecanase-l secondarily cleaves at the same site as MMPS (Westling et a1 2002). The majority of aggrecan fragments found in OA synovial fluid are derived from cleavage due to aggrecanases (Sandy et al 2001). Although MMP 3 and 13 degrade the interglobular domain (IGD) of aggrecan, increased MMP expression is not correlated with GAG release into media of explant cultures (Little et a1 1999), in contrast to a documented correlation with aggrecanase activity (Westling et al 2002). MMPS may play an important role in the later stages of aggrecan degradation through primary cleavage of the IGD and secondarily through action on aggrecanase generated metabolites (Little et a1 2002). 21 ADAMTS 4 and 5 activity is detected in joint capsule, synovium and cartilage and may be upregulated in OA synovium either transcriptionally or post-translationally (Amer 2002). Results are conflicting as to the relative importance of each aggrecanase in OA. Whilst aggrecanase-2 expression, but not aggrecanase-1, increases in response to IL-1 stimulation of human chondrocytes (Koshy et al 2002), aggrecanase-1 expression predominated in another study (Bau 2002). Tissue Inhibitors of Metalloprateinases The tissue inhibitors of metalloproteinases (TIMPS) are the major endogenous regulators of the MMPS (Nagase et al 2003). In normal cartilage, TIMPS exist in slightly greater concentration than MMPS (Dean et al 1987), with aberrant MMP expression a feature of 0A. TIMPS are constitutively expressed by synovial fibroblasts, and also produced by chondrocytes. Currently 4 isoforrns have been identified, designated TIMP 1, 2, 3 and 4 (Brew et al 2000). TIMPS bind to MMPS in a 1:1 stoichiometry, and inhibit all MMPS except MMP 14. Equine synovial fluid contains both TIMP 1 and TIMP 2 with increased levels in OA joints (Clegg et al 1998a). In OA, both synovial and cartilage TIMP l and 3 mRNA levels are elevated (Su et al 1999). TIMPS bind to MMPS with varying degrees of affinity. TIMP 3 is a relatively weak inhibitor of MMP 3, with similar inhibition of MMP l and MMP 2 as TIMP 2 (Kashiwagi et al 2001). In addition, TIMP 3 binds to sulfated GAGS (Yu et a1 2000), and inhibits aggrecanases, for which it has a stronger affinity than for MMP 1, 2 and 3 (Kashiwagi et al 2001). TIMP 3 binds to the active binding site of aggrecanases (Kashiwagi et al 2001), whilst TIMP 1 can inhibit activation of 22 aggrecanase-1 (Gao 2002). The ability of TIMP 3 to bind to the ECM is thought to be important for localized regulation of MMP activity. Inflammatory Mediators Nitric oxide Nitric oxide is a cytotoxic free radical, the by-product of oxygenation of L-arginine. This conversion is catalyzed by nitric oxide synthase (NOS), of which there are 3 isoforms: NOS I and III are constitutively expressed whereas NOS II (iNOS) is inducible and transcriptionally regulated. Chondrocytes, particularly in superficial cartilage layers (Hayashi et a1 1997), are the major source of iNOS (Grabowski et al 1997), with spontaneous production of NO in OA and enhanced production with cytokine stimulation (Pelletier et al 1996). NO induces a number of pathophysiological events characteristic of OA including enhanced MMP synthesis (Murrell et a1 1995), and reduced IL-lRa (Pelletier et al 1996), PG (Oh et al 1998), and type II collagen synthesis (Cao et al 1997). IL-IB is a potent inducer of iNOS activity in chondroytes (Frean et a1 1997). Selective inhibition of iNOS in a canine model of OA decreased MMP, IL-IB and COX 2 expression (Pelletier et al 1999). In equine articular cartilage explants, NO mediated the suppressive effect of IL-1 on PG synthesis (Bird et a1 2000), and inhibited aggrecan degradation, suggestive of a possible anti-catabolic effect of NO. Production of NO was increased in explants obtained from GA compared to normal equine cartilage. Further, 23 NO synthesis by equine articular cartilage is consistently higher than that of synovial membrane (von Rechenberg et al 2000), with similar results obtained in a canine cruciate ligament rupture model of OA (Spreng et a1 2000) IL-l appears to mediate activation of iNOS via distinct signaling pathways. Stimulation with IL-1 induced a time and dose dependent increase in iNOS mRNA synthesis and activity, along with induction of NF KB and AP-l (Mendes et al 2002). In addition, the degradation of the inhibitor of kappa B (Icha) was inhibited as was NFKB DNA binding activity. This suggests an autoregulatory effect, consistent with previous findings that NO could modulate its own production by interfering with the interaction of NFKB with its binding Site in the promoter region of the iNOS gene (Park et a1 1997). Regulation may also be effected by reduction in iNOS catalytic activity (Colasanti et al 1995). Pathways appear to be diverse and complex with Significantly decreased levels of both IL-1- converting enzyme (ICE) and IL-18 following selective inhibiton of iNOS (Boileau et al 2002). Further, additional reactive oxygen Species such as superoxide react with NO to form peroxynitrite and may be required for IL-1 induced IKBO. degeneration and consequently NFKB activation and iNOS expression (Del Carlo et a1 2002, Mendes et a1 2003) Prostaglandins Prostaglandins, especially PGEz, are produced in inflamed joints, and increased synovial fluid PGE2 concentration occurs in OA (Gibson et al 1996). Equine synovial membrane 24 PGEz concentration may exceed that of articular cartilage, with highest levels seen in moderate OA (von Rechenberg et al 2000). Spontaneous production of PGE2 by OA cartilage has been related to up regulation of COX 2 activity (Amin et al 1997). Actions of PGE2 in joints include vasodilation, cartilage proteoglycan depletion and enhanced pain perception. At least some elements of pain in joints may be mediated by substance P, a neurotransmitter found in increased concentration in equine 0A synovial fluid (Caron et al 1992), and the concentrations of which are associated with PGE; levels (Kirker-Head et al 2000). In equine monolayer cultures, exogenous PGEz had no effect on resting MMP and TIMP gene expression, but significantly decreased IL-l induced gene expression of MMP 1, 3 and 13 as well as TIMP 1, suggesting a potential role for prostanoids in MMP regulation (Tung et al 2002a). In further support of this role, PGEZ also inhibited rhIL-IB induced stimulation of MMP and TIMP 1 mRNA expression in human synovial fibroblasts (DiBattista et al 1995). The Role of Cytokines A number of studies have examined the effects of cytokines on cellular metabolism and their role in the pathophysiology of OA (Martel-Pelletier et al 1999). Cytokines appear to be first produced by cells of the synovial membrane (Pelletier et a1 1995), and later by activated chondrocytes (Pelletier et al 1993). The predominant cytokines synthesized in 0A are interleukins and tumor necrosis factor (TNFor), with IL-1 being the primary mediator of the characteristic degradative processes. IL-1 and TNFor can induce further 25 cytokine production by synoviocytes and chondrocytes, including enhanced expression of not only these cytokines but also IL-8, IL—6, leukemia inhibitory factor and proteinases. TNF-a Tumor necrosis factor exists as two forms, TNF-or and TNF-[3. TNF-or induces synovial membrane inflammation and plays a role in degradation of the ECM. TNF-or, synthesized as a precursor protein, is activated via proteolytic cleavage by TNF-or converting enzyme (TACE) (Martel-Pelletier et a1 1999). Levels of both TNF-or and TACE mRNA are increased in OA compared to normal cartilage (Femandes et al 2002). Two types of TNF receptors are present on the cell membrane surface of most tissues (TNF-R55 and -75). In OA, chondrocytes and synovial fibroblasts have enhanced expression of TNF-R55. OA synovial fibroblasts and chondrocytes also spontaneously produce two soluble receptors, especially TNF-SR75 (Alaaeddine et al 1997). Interleukin-I Two forms of IL-1, IL-lor and IL-IB, have been identified. Of the two forms, IL-lB has received the most attention. It is synthesized as a precursor, and converted to its active form by the serine protease, IL-IB converting enzyme (ICE), prior to its release (Martel- Pelletier et al 1999). Both forms mediate their effects on cells through a cell membrane associated receptor (IL-1R). The two forms of this receptor are type I and type II (IL-1R1 and IL-lRII), with the type I receptor having a higher affinity for IL-IB. The expression of IL-1 receptors, particularly type I (Sadouk et al 1995), by chondrocytes and synovial fibroblasts increases in OA, resulting in cells that are more sensitive to stimulation by IL- 26 1. Both types of IL-lR can be shed extracellularly in a soluble form (IL-15R). Because ligand-binding regions are preserved, these soluble receptors are thought to function as receptor antagonists (F emandes et a1 2002). A receptor-associated protein (ILl-RAcP) is required as a co-receptor for IL-1 signaling (Radons et al 2002). The response to IL-1 stimulation in vitro is multifactorial and includes synthesis of phospholipase A2, COX 2, PGE2, thromboxane A2 and iNOS; and inhibition of collagen synthesis (type 11, IX, and XI) (Cook et al 2001, Murrell et al 1995, Tung et al 2002b). IL-l also increases the induction and secretion of MMPS (Caron er al 1996b, Reboul et a1 1996, Richardson et al 2000), GAG release into media and aggrecanase activity (Cawston et al 1999). The effect on TIMPS appears to be minimal in some studies (Cawston et al 1999) with conflicting results in others, with both downregulation (Sadowski et al 2001) and upregulation reported (Tung et al 2002b). The effect on PG homeostasis appears two-fold, with dose dependent induction of PG degradation (Frean et a1 2000) and inhibition of PG synthesis (Monis et al 1994, Frisbie et a1 1997, MacDonald et a1 1992, Platt et al 1994). Elevated levels of IL-l-like biological activity in the synovial fluid of horses with clinical OA supports the role of IL-1 in degenerating equine joints (Morris et a1 1990, Alwan er a1 1991). Further, IL-IB has been detected in human OA synovial fluid (Kahle et a1 1992). Pharmacological inhibition of the effects of IL-1 on equine cartilage, as a means to slow or arrest OA progression, illustrates its importance in OA (Caron et al 1996a, Frisbie et a1 2000, Frean et012000). 27 Early in vitro studies utilized recombinant human interleukin-1 (rhIL-l), and demonstrated inhibition of ECM synthesis and induction of ECM depletion. In human chondrocyte cultures, rhIL-IB suppressed synthesis of type II collagen, associated with decreased (11(11) procollagen mRNA levels (Goldring et al 1998). Aggrecan mRNA also down regulated following exposure to IL-1 (Richardson et a1 2000). Equine chondrocytes in explant and monolayer culture stimulated with rhIL-l synthesize MMPS (Morris et al 1994, Caron et al 1996b) and the ECM of cartilage explants degrades as evidenced by decreased GAG and PG synthesis (MacDonald et al 1992, Frisbie et al 2000). Coincident with enhanced cartilage matrix MMP activity (including MMP 1, 13 and 3), are augmented concentrations of other inflammatory mediators such as PGE2 and NO. Stimulation with rhIL-lor in equine explant cultures dose dependently decreased MMP 3, GAG and PG synthesis; with a compensatory increase in PG synthesis seen once stimulation was discontinued (Morris et al 1994). The response to rhIL-l stimulation varies, with an overall reduced response to IL-1 in cartilage obtained from older humans and horses (Ismaiel et a1 1992, May et al 1992b, Morris et al 1994, MacDonald et al 1992). The effect of age was significant in terms of GAG synthesis, with greater change for a given increase in IL-1 concentration in cartilage obtained from younger horses, demonstrating a Significant dose-by-age interaction (MacDonald et al 1992). Although PG synthesis was inhibited in all age groups, IL-l did not influence degradation of PG in mature equine cartilage (Platt et al 1994). 28 The response to IL-1 varies between species and with the source of IL-1. A partially purified form of equine IL-l obtained from mononuclear cells following LPS stimulation resulted in dissimilar and disproportionate stimulation of PGE2 and MMP synthesis by chondrocytes and synoviocytes compared to rhIL-l (May et al 1990). In vitro, activity of rhIL-l B is greater than that of rhIL-la (May et al 1992a), attributable to differing affinity of IL-1 receptors on equine cells for the different forms of rhIL-l (May et al 1992b). Similarity between equine and human amino acid sequences of IL-1 [3 is only 26% (Howard et al 1998). Given this lack of Similarity, it is possible that the response of equine tissue to rhIL-lB may not be entirely representative of the in viva Situation. This finds support in research documenting that although only 5% of receptors need to be occupied for half maximal stirnution of MMPS by rhIL-IB in human synoviocytes and chondrocytes (Martel-Pelletier et a1 1992), 2-3 times this concentration is required for receptor saturation in other species (Chin et al 1990). Such variability and a need to assure species-specific effects prompted the development of a species- specific IL-IB (reIL-113) (Tung et al 2002b). The nucleotide and deduced amino acid sequence homology of the species specific reIL with human and murine IL-lor was 71.6% and 60.2%, and 66.7% & 61.8% for IL-IB (Kato et al 1995). Purified reIL-10 in equine monolayers lead to dose saturable up regulation of MMP 1, 3, 13, TIMPl, and COX 2 gene expression (Tung et al 2002b), along with enhanced MMP activity and nitrite concentration in media. In equine cartilage explants (Takafuji et al 2002) both reIL-1a and reIL-1B (0.1- lOOng/ml) dose dependently increased PGE2 synthesis and PG release into media and 29 decreased PG synthesis, in agreement with previous studies utilizing rhIL-l that had demonstrated near maximal response at concentrations greater than or equal to 0.1 ng/ml (MacDonald et a1 1992, Platt et al 1994, Morris et a1 1994). However, concentrations of reIL-1 were 40-100 times lower than concentrations of rhIL-l used in previous studies had substantial effects on PG degradation and synthesis. The interleukin-1 receptor antagonist (IL-lRa) is a competitive inhibitor of IL-IR. IL- lRa is capable of inhibiting a number of pathological events in OA including synoviocyte PGE2 synthesis, chondrocyte collagenase production, and degradation of ECM (Pelletier et al 1999). There are three forms of IL-lRa: soluble IL-lRa (IL-lsRa), and two intracellular forms (icIL-lRaI and icIL-lRaII) (Arend 1993). Both IL-lsRa and icIL-lRa can bind to IL-lR, with the soluble form binding with greater affinity. A relative deficiency of IL-lRa occurs in 0A. In dogs with experimentally induced OA, intra- articular injection of IL-lRa not only reduced MMP 1 mRNA expression, but also resulted in a dose dependent protective effect on osteophyte development along with a reduction in severity of cartilage lesions (Caron et al 1996a). The potential for using IL- lRa as a clinically applicable treatment for CA is still being investigated. Other Cytokines In addition to TNF-or and IL-10, a number of other cytokines may play a role in OA. Stimulation of chondrocytes with IL-1 induces IL-8 synthesis, which in turn stimulates the release of superoxide radicals and lysosomal enzymes from chondocytes (Platt 1996). 30 Although the exact role of IL-8 in ECM degradation is unknown, it is a potent chemotactic factor for neutrophils and as such may influence inflammatory cell migration. IL-6 is constitutively produced in human chondrocytes, with a dose dependent increase following stimulation by IL-1, TNFor and LPS (Bunning et al 1990). IL-6 amplifies the effects of IL-1 on MMP synthesis and proteoglycan synthesis (Nietfield et al 1990); however its role in 0A is as yet unclear, as IL-6 induced TIMP production (Lotz et al 1991). IL-17 also influences chondrocytes metabolism. Specifically, it up regulates IL-IB, TNFa, and IL-6 in a number of cell types, and increases NO production in chondrocytes (Attur et al 1997, Martel-Pelletier et al 1999). Leukemia inhibitory factor (LIF) is a glycoprotein found in increased concentration in synovial fluid of OA patients (Dechanet et a1 1994). LIF enhances IL-IB expression in chondrocytes and synovial fibroblasts (V illiger et al 1993), and stimulates the production of NO (Reid et al 1990). In contrast, IL-11 in articular chondrocytes and synovial fibroblasts has no effect on MMP production, but rather induces TIMP synthesis (Maier et al 1993) and decreases PGE2 release by DA synovial fibroblasts (Alaaeddine et al 1999). IL-4, IL- 10 and IL-13 have anti-inflammatory effects. IL-4 and IL-10 inhibits synthesis of IL-1 and TNF-or, whereas addition of IL-13 increases IL-lRa production (Femandes et a1 2002) 31 Interleukin-1 Signaling Path ways Following binding of IL-1 to its cell associated receptor, a number of phosphorylation dependent Signaling pathways are initiated, leading to the production of transcription factors that subsequently regulate gene expression. The two main pathways by which this occurs involve the transcription factors AP-l and NFKB. The initial portions of their signaling pathways are shared. After IL-l binds to its receptor (typically type I receptor for IL-IB), additional binding to the IL-1 receptor accessory protein (IL-lRacP) is required for signal transduction (Figure 1). Binding of IL-1 to its receptor induces conformational changes and recruitment of multiple receptor-bound proteins, including myeloid differentiation protein (MyD88). MyD88 functions act as an adaptor protein, coupling activation of the receptor to downstream Signaling components (O’Neill et al 2003, Takeuchi et al 2002). MyD88 contains a Toll/Interleukin-l receptor domain (TIR) through which it interacts with the IL—lR (Burns et al 1998). Subsequently, IL-1 receptor associated kinase (IRAK), a serine/threonine kinase, is recruited to the receptor complex by binding to MyD88. The Toll-interacting protein (Tollip) is involved in IRAK recruitment through association with IL-lRacP (Burns et a1 2000). IRAK is autophosphorylated and then dissociates to interact with another adaptor, tumor necrosis factor receptor associated factor 6 (TRAF6), recruited to the receptor complex upon phosphorylation of IRAK (J iang et al 2002). In turn, TRAF6 once ubiquitinated, activates pathways that ultimately results in the formation or activation of AP—l or NFKB. 32 Activator Protein-1 Activator Protein-1 is a transcription factor composed of two proteins of the Jun and F 03 families. Its most common form is the heterodimer c-Jun/c-Fos. AP-l is one of the end products of the mitogen activated protein kinase (MAPK) Signaling pathways. The MAPKS include the serine/threonine kinases such as c-Jun N terminal kinase (JNK), extracellular Signal-related kinases (ERK), and p38, all of which are activated by IL-1 in chondrocytes (Geng et a1 1996). The MAPKS are at the downstream end of a 3-tiered system that also contains mitogen activated protein kinase kinases (MAP2K) and mitogen activated protein kinase kinase kinases (MAP3K). Following activation (Figure 1), TRAF6 associates with the TRAF associated protein evolutionary conserved signaling intermediate in Toll pathways (ECSIT) (Kopp et al 1999). ECSIT then interacts with mitogen activated protein kinase/extracellular signal- regulated kinase kinase kinase-1 (MEKK-l), a MAP3K, linking TRAF6 to the MAPK pathway. Subsequently, MEKK-l activates a MAP2K that then activates the MAPK JN K (Ninomiya-Tsuji et a1 1999). JNK phosphorylates c-jun at two N-terminal serines in its transactivation domain, providing the primary mode of c-jun regulation. Two JNK enzymes have been identified, JNK-1 and JNK-2 (also called MAPK9). JNK-2 through its higher binding affinity for c-jun is probably the more physiologically relevant form (Firestein et al 1999). Following activation, c-jun translocates to the nucleus to interact with c-Fos, thereby forming AP-l. AP-l then binds to the promoter region of a number of genes, including 33 MMPS, to influence their transcription. The role of transcription factors such as AP-l in the response of synovium and chondrocytes to IL stimulation was originally investigated in models of rheumatoid arthritis. These studies established a key role of AP-l and localized its components to synovium, with constitutive expression of JNK-2 by RA synoviocytes. Patients with rheumatoid arthritis have higher AP-l binding activity compared to patients with GA (Asahara et al 1997). However, the pattern of MAPK activation in synoviocytes may differ to that in chondrocytes, and patterns differ depending on the initial stimulus. IL—l stimulation of chondrocytes activates JNK-1 and — 2, ERK and p38 in a time dependent manner. Conversely, in response to LPS stimulation only ERK is stimulated (Geng et a1 1996). Nuclear F actor-KB NF KB is a ubiquitous protein that exists in the cytoplasm in inactive form, bound to its inhibitory subunit 1ch (inhibitor of KB). The most prevalent activated form of NFKB is a heterodimer of p50 and p65 subunits that contain transactivation domains necessary for gene induction (Tak et al 2001). Following activation of TRAF6 (Figure 1), TRAF6 dissociates and translocates to the cytoplasm to form a complex with transforming-growth-factor-B-activated kinase-l (TAKl), and transforming growth factor B activated Kinase-l binding proteins 1 and 2 (TABl and TAB2) (Qian et al 2001). TAKl, a MAP3K, plays a critical role in IL-1 mediated activation of the NFKB pathway by phosphorylating and hence activating NF- KB-inducing kinase (NIK) (Takaesu et a1 2003). TAB2 is membrane bound, and 34 translocates to the cytoplasm when stimulated by IL-1 to function as an adaptor linking TRAF 6 to TAKl and TABl (Qian et al 2001). In turn, NIK activates inhibitor of kappaB kinase (IKK) that is responsible for the phosphorylation of IKB. Phosphorylation of IKB leads to ubiquitination with subsequent degradation by proteosomes, thereby releasing NFKB. NFKB subsequently translocates to the nucleus where it binds to KB enhancer elements in the promoter region of a number of genes, thereby activating their transcription (Tak et al 2001). IKK contains two subunits, or and B, as well as a regulatory subunit (IKKy). IKKor is involved in transient NFKB activation while IKKB is involved in sustained activation (Tak et a1 2001). IKK activity increased ten fold within ten minutes of IL-1 stimulation of synovial fibroblasts, with increased IKK activity preceding NF KB translocation to the nucleus (Firestein et al 1999). IKK resides at a key convergence Site for multiple signaling pathways that lead to NFKB activation. The primary pathway by which proinflammatory stimuli such as IL-1 and TNFa induce NFKB firnction is via activation of IKK-B rather than IKK-or (Aupperle et al 1999). Although both IKK-or and -B are constitutively expressed, production is enhanced by IL-1. NIK can also activate NFKB through association with another TRAF, TRAF 2 (Firestein et al 1999). NFKB binding sites are present on the promoter regions of a number of inflammatory genes, including IL-1, IL-2, IL-6, IL-8, TNFoc, MMPS 1,3,9,13, iNOS, and COX 2 (Baldwin 2001, Baeuerle et al 1997, Mengshol et a1 2000, Tak et al 2001). IL-l induced 35 collagenase expression in synoviocytes is primarily activated by the p50 homodimer that binds to a critical NFKB-like binding site (V incenti et a1 1998). NFKB in synoviocytes has an antiapoptotic role and plays an important function in protection against cytotoxicity of TNFor (Miagkov et a1 1998). AP-l and NFKB pathways appear to diverge at the level of IRAK/TRAP 6 (Li et al 2001). However, these pathways are complex with cross-talk possible between signaling components. TAKl can activate NIK and hence NFKB, as well as JNK (Takaesu et a1 2003). More than one IRAK is involved in IL-1 signaling, and that alternative splicing of adaptor proteins such as MyD88, may influence the ultimate end product of these pathways. Myd88 acts as a bridging protein between IRAK-1 and IRAK-4, enabling IRAK-4 induced IRAK-1 phosphorylation (Burns et a1 2003). The alternative Splice form of Myd88 (MyD883) may prevent NF KB activation, by preventing recruitment of IRAK- 4 and hence phosporylation of IRAKl , while still allowing for phosphorylation and hence activation of JN K (Janssens et al 2003). Role of AP-l and NFKB in 0A The role of both AP-l and NF KB in CA has been investigated, building largely on results obtained from in vitro studies of rheumatoid arthritis (RA) as well as other cell types. In both RA and OA, NFKB expression is increased concurrent with increased AP-l components, c-Jun and c-Fos. DNA binding activities of both AP-l and NFKB are greater in RA than OA, with elevated expression preceding the development of clinical signs and increased MMP levels (Han et al 1998). 36 Following IL-l stimulation, the effects of COX 2, an enzyme known to have a crucial role in OA, are mediated by NFKB. COX 2 has two putative NFKB Sites in its 5’ promoter (Newton et al 1997). Other transcription factors, such as the p38 MAPK, have also been implicated in the regulation of COX 2 gene expression in chondrocytes (Thomas et a1 2002). NFKB and AP-l are early response genes required for the transcription of MMPS following IL-l stimulation (V incenti et al 2001). The role of AP-l in chondrocytes and synoviocytes is mediated via JNK (Mengshol et al 2000, Han et al 2001). The promoters of MMPS such as MMP 1 and 13 contain a core transcriptional unit (TAT box) at approximately -30 base pairs and an AP-l binding site at —70 base pairs (Borghaei et a1 1997). The latter is a TGAG/CTCA sequence that binds dimers of c-fos and c-Jun (Vincenti et a1 2002). These binding sites correlate with inducible MMP expression (Borden et al 1997); however, several other AP-l sites throughout MMP promoters may also contribute to gene expression (Benbow et al 1997). In murine models of inflammatory arthritis, activation of AP-l and NFKB preceded clinical signs and MMP 1 and MMP 13 gene expression (Han et al 1998). The MMP13 promoter region contains a conserved AP-l binding site that binds c-Jun and c-Fos (Tardif et al 1997), and transcription of AP-l increases prior to the induction of MMP 13 gene expression (Goldring et al 1994). 37 Interestingly, IL-l induction of MMP 13 in chondrocytes requires both NFKB and JNK (Mengshol et a1 2001). These cells rely on p38, JNK and NFKB to activate MMP l3 (Mengshol et al 2000), suggesting that the MAPKS either directly or indirectly activate AP-l subunits that then cooperate with NFKB to activate MMPS. AP-l sites may interact with NFKB elements that are also present in MMP promoters (Barchowsky et al 2000, Vincenti et al 1998). Both AP-l and NFKB binding sites are required for MMP 1, 3 and 9 expression (Bond et al 1998, Vincenti et a1 1998); however, JNK or NFKB alone may be sufficient to increase MMP 1 expression (Bond et a1 1999). Drugs used in Osteoarthritis Treatment Nonsteroidal anti-inflammatories Nonsteroidal anti-inflammatories (N SAIDS) are frequently used to treat musculoskeletal disease, including OA, in horses. Commonly used NSAIDS include phenylbutazone and flunixin meglumine. NSAIDS are cyclooxygenase inhibitors, and thereby inhibit the production of prostaglandins. NSAIDS reduce the clinical signs of OA such as pain and synovitis, associated with reduced synovial fluid PGE2 concentration (Owens et al 1996). Concerns regarding the use of NSAIDS mainly relate to their potential side effects such as gastric ulceration, right dorsal colitis and renal papillary necrosis and medullary ischemia (Moses et a1 2002). In addition, some NSAIDS have negative effects on both chondrocytes and bone, including inhibition of GAG synthesis (Dingle 1999), decreased mineral apposition rate in cortical bone and reduced healing rate of experimentally induced cortical defects (Rohde et al 2000). Clinical trials of the effect of oral 38 phenylbutazone (4.4mg/kg BID for 14 days) on cartilage metabolism showed a significant reduction in PG synthesis (Beluchi et al 2001). A number of in vitro studies have been conducted to elucidate both efficacy and potential mechanisms of action of NSAID in 0A. The reported effects of different NSAIDS on articular cartilage vary. In some studies, NSAIDS reduced PG catabolism and MMP activity induced by IL-1 (Pelletier- et a1 1999). Indomethacin inhibits MMP 3 and increases TIMP 1 expression (Yamada et al 1996), while phenylbutazone also inhibits MMP 3 synthesis, albeit at concentrations that exceed those achieved therapeutically (May et al 1988). Conversely, in another study, flunixin meglumine and phenylbutazone had no effect on MMP expression (Clegg et a1 1998). NSAIDS increased PG synthesis in cartilage explants (Jolly et a] 1995), and PGE2 synthesis decreased in equine explants; with no effect on COX 2 or iNOS gene expression (Tung et al 2002c, d). Carprofen attenuated the LPS induced increase in IL-6 in equine chondrocytes and synoviocytes, but had no effect on release of IL-1 (Armstrong et a1 2002). Similar to their effects in cartilage, NSAIDS suppressed PGE2 production in equine synovial membrane explants without detrimental effects on synovial membrane viability and function (Moses et a1 2001). NSAIDS are capable of influencing some of the aforementioned IL-l signaling pathways. Specifically, both aspirin and sodium salicylate block IKKB, thereby preventing NFKB activation of genes including COX 2 (Yin et a1 1998). The influence of phenylbutazone 39 and flunxin meglumine, the most commonly used NSAIDS in equine practice, remains to be determined. Corticosteroids Inna-articular administration of corticosteroids in horses with joint disease is widespread. Corticosteroids inhibit phospholipase A2; however, their anti-inflammatory effects extend beyond inhibition of arachidonic acid metabolites. Corticosteroids diffuse into the cytoplasm to interact with steroid-specific receptors, and subsequently bind to promoter regions of glucocorticoid-responsive genes, thereby modulating their transcription (Trotter 1996). In vitro, corticosteroids inhibit a variety of degradative enzymes, including MMP 13 (Caron et al 1996b), COX 2 and PGE2 (Tung et a1 2002d). MMP 1, 3, 13 and TIMP l are inhibited with lower doses than those required for down-regulation of type II collagen and aggrecan gene expression in equine chondrocytes (Richardson et al 2003). Dexamethasone treatment results in pretranslational regulation of iNOS expression in vitro (Tung et al 2002c). Dexamethasone decreases PG content more than that induced by IL-1 stimulation alone (Frisbie et al 1997, Stove et al 2002). In vivo, corticosteroids reduce the progression of experimentally induced OA lesions (Pelletier et al 1995). The action of corticosteroids is due in part to their effects on IL-1 signaling pathways. The glucocorticoid-receptor complex binds to AP-l (Krane 1993), and induction of the IKBoc inhibitory protein leads to inhibition of NFKB (Auphan 1995, Kovalovsky et a1 40 2000). The glucocorticoid-receptor complex can also repress NFKB through physical interaction with p65 subunits to inhibit NFKB transcription (Scheinmann et a1 1995). Hyaluronan Sodium hyaluronan (HA) is frequently used in the treatment of equine joint disease (McIlwraith et al 1997). The specific mode of action is yet to be determined; however, HA improved mobility and reduced pain in OA joints (Ghosh 1994). HA may be administered intra-articularly or systemically. A number of clinical trials documented an improvement in lameness score (Gaustad et al 1995), as well as decreased synovial fluid total protein and PGE2 levels (Kawcak et al 1997). Following intra-articular administration, the clearance of HA is fairly rapid (Hilbert et a1 1995); however, a portion remains associated with synovial tissues. In humans, intra-articular HA is frequently used to treat pain associated with OA, with proven efficacy and safety (Altman et a1 1998). In a rabbit instability model of OA, once weekly intra-articular injections of HA reduced disease progression and improved morphological appearance of articular cartilage (Amiel et al 2003). In human and animal in vitro OA models, HA exerted effects on both synovium and cartilage, including stimulation of synoviocyte HA production (Smith et al 1987), prevention of PG and collagen degradation (Takahashi et a1 1999, Morris et al 1992), stimulation of PG synthesis (Frean er a1 1999), prevention of chondrocyte apoptosis (Takahashi et al 2000), and reduction of inflammatory mediators such as NO (Punzi 2001). HA also inhibits IL-l induced PGE2 production by chondrocytes (Akatsuka 1993), 41 and synoviocytes (F rean et a] 2000). HA appears to exert no effect on COX 2, iNOS (Tung et al 2002a, b) or MMP expression (Clegg et al 1998, May et al 1988). Some favourable effects of HA may be in part due to inhibition of leukocyte migration and function (Howard et al 1996), as well as inhibition of arachidonic acid release from membrane phospholipids of synovial fibroblasts (Tobetto et al 1992). Polysulfated Glycosaminoglycans Polysulfated glycosaminoglycans (PSGAG) such as Adequan®, are semisynthetic heparinoids. The principal GAG in this putative “chondroprotective” agent is chondroitin sulfate. Inna-articular administration of PSGAG is thought to be of benefit in the treatment of lameness (Hamrn et al 1984, Caron et al 1996c). It appears to be more effective when given intra-articularly rather than following intramuscular administration (Trotter et al 1989). Unfortunately, side effects of intra-articular administration of PSGAG have been documented including flare reactions (Todhunter et a1 1994) and possible sepsis. PSGAG increased MIC of antibiotics required in vitro against Staphylococcus aureus and dramatically reduced the intra-articular inoculum of this bacterium to induce sepsis in vivo (Gustafson et al 1989a, b). The precise mechanism of action of PSGAG is unknown. It has an affinity for proteoglycans and non-collagenous proteins, and stabilizes fibronectin-collagen complexes (Andrews et al 1985). PSGAG inhibits a variety of degradative enzymes, including MMP 3 (May et a1 1988) and MMP 2 and 9; however, the latter effect is only recognized at concentrations exceeding those achieved clinically (Clegg et a1 1998). 42 After experimental induction of 0CD lesions in horses, PSGAG decreased type II collagen in articular cartilage (Todhunter et al 1993); however, in other studies, collagen and GAG synthesis were stimulated (Glade 1990). The reported effects of PSGAG on PG synthesis and degradation vary. A protective effect on IL-1 induced inhibition of PG synthesis was observed (Frean et al 2002). A surmised stimulatory effect on PG synthesis in normal and 0A cartilage was not demonstratable, and minimal effects on PG degradation were observed in unstimulated cultures (Caron et al 1991 and 1993). PSGAG had no effect on synthesis and release of PGE2 by reIL-10 stimulated equine chondrocytes or on COX 2 gene expression; however, iNOS expression decreased (Tung et al 2002a, b). Pentosan Polysulfate Pentosan Polysulfate (PPS) is a polysulfated polyglycosarninoglycan heparin analogue derived from beech wood hemicellulose. Pentosan polysulfate is available in two forms, a sodium salt and a calcium derivative. In vitro, effects have included increased TIMP 3 synthesis (Takizawa et a1 2000), inhibition of aggrecan degradation (Munteanu et al 2002), increased synovial fluid hyaluronan (Francis et al 1993), stimulation of PG synthesis, and inhibition of MMP 3, but not MMP 2 and 9, synthesis (Rogachefsky et al 1993, Clegg et al 1998). Pentosan polysulfate binds to and is internalized by synoviocytes and chondrocytes (Ghosh et al 1996). Further, PPS inhibited aggrecanase mediated degradation of aggrecan in bovine explant cultures (Munteanu et al 2002). The anti-thrombotic effects of PPS are thought to improve subchondral bone blood flow (Ghosh 1999). 43 Although pharmacokinetic studies are limited, therapeutic plasma and synovial fluid concentrations have been detected following single intramuscular administration of calcium PPS (2mg/kg) in the horse (Fuller et a1 2002). The efficacy of oral calcium PPS (10mg/kg) was evaluated in a double blind placebo controlled study in dogs with cranial cruciate ligament injury (Innes et al 2000). Functional outcome and radiographic progression were unaltered; however, reduced ECM breakdown was inferred from a significant decrease in 5D4 epitope of keratan sulfate in synovial fluid. In a placebo controlled, blinded clinical study in horses, sodium PPS (3mg/kg) improved lameness and flexion scores, reduced effusion and synovial fluid total protein, reduced radiographically evident entheseophyte production and subchondral bone lysis, and reduced cartilage erosion and fibrillation (Frisbie et a1 2003). However, serum markers of cartilage breakdown were increased (carboxy propeptides of type II procollagen and epitope 846 of chondroitin sulfate). PPS had no effect on clotting factors or platelets. However, in a study investigating the effects of sodium PPS on hematological and hemostatic values in horses, activated partial thromboplastin time was dose dependently increased (Dart et a1 2001). Glucosamine and Chondroitin sulfate Structure Glucosamine and chondroitin sulfate have been classified as both “nutraceuticals” and Symptomatic Slow Acting Drugs In Osteoarthritis (SYSADOA). Glucosamine is an 44 amino monosaccharide (2-amino-2-deoxy-alpha-D-glucose) consisting of a hexose sugar ring (glucose) with an amino group substituted for a hydroxyl group on carbon 2. Glucosamine, once modified as N-acetylglucosamine, is a precursor of the disaccharide units of GAGS such as HA and keratan sulfate. Isomerisation converts glucosamine to galactosamine, a structural component of chondroitin sulfate and derrnatan sulfate. Most glucosamine in the body is in the form of glucosamine-6-phosphate (Platt 2001). Glucosamine is commercially available in three forms: glucosamine hydrochloride (GHCl), glucosamine sulfate (GS) and N-acetyl-D-glucosamine (NAG). Chondroitin sulfate is a GAG consisting of alternating units of glucuronic acid and sulfated N—acetyl galactosamine. The two forms of chondroitin sulfate vary in the position of the sulfate residue attached to N-acetylgalactosamine (C-4-S and C-6-S). Actions in Articular Tissues Glucosamine Initially, beneficial effects of glucosamine supplementation were attributed to the provision of raw materials required for “building blocks” of cartilage, namely GAGS. Theoretically, by supplying an exogenous form of glucosamine, the rate-limiting step of synthesis of glucosamine-6-phosphate is bypassed, thus increasing the rate of HA synthesis (Kim 1974). Exogenous glucosamine is preferentially utilized in the synthesis of GAGS when cells are cultured without glucose (Roden 1956). The preferential incorporation of glucosamine into galactosarnine moieties of CS in articular cartilage explants supported the use of glucosamine as a source of cartilage matrix components 45 (Noyszewski et al 2001); however, this mode of action has been debated (Mroz et a1 2004) Glucosamine enters chondrocytes via facilitated glucose transporters (GLUT), acting as a competitive inhibitor of glucose transport (Windhaber et a1 2003). Glucosamine has a greater affinity for certain glucose transporters, especially GLUT2, in other cell types (Uldry et al 2002); however, in chondrocytes, glucose uptake is non-insulin dependent as GLUT4, the major insulin regulating glucose transporter, has not been identified in cartilage (Shikhman et a1 2004). Potential chondroprotective effects include stimulation of GAG and PG production by GS (10-100ug/ml) in human 0A chondrocytes in vitro, with a carry over effect evident for at least 4 days after the withdrawal of glucosamine from media (Bassleer et al 1998). Glucosamine is thought to stimulate the synthesis of aggrecan (Jimenez et al 1997, Dodge et al 2003). Glucosamine appears to have no influence on type II collagen production (Bassleer et a1 1998, Dodge et al 2003). The lack of effect on collagen synthesis was also documented in a rabbit chymopapain induced 0A model, despite a significant increase in GAG content of both affected and normal cartilage in the contra- lateral limb (Oegema et al 2002). The effect of glucosamine on PG synthesis may be attributable to an increase in GAG precursors such as UDP-N-acetylglucosamine and galactosarnine (Sandy et al 1998, Patwari et a1 2000), or it may act as a direct substrate of glycosyltransferases, enzymes 46 involved in post-translational modification of GAGS. Glucosamine prevented IL-l induced repression of galactose B-l,3-glucuronosyltransferase I (GchT-l), a key enzyme that catalyzes the addition of the first glucuronic acid residue to the trisaccharide backbone of GAGS (Gouze et al 2001). Hence, exogenous glucosamine could, by supplying precursors for the glycosyltransferases, bypass glutamine fructose-l-phosphate transaminase, the rate-limiting enzyme in the hexosamine pathway. Further contribution to GAG synthesis may be through promotion of incorporation of sulfur into cartilage, as administration of GS increased both serum and synovial fluid sulfate concentrations (Hoffer et al 2001). Glucosamine also has an anti-inflarnmatory activity, suppressing inflammatory mediators and degradative enzymes such as NO, PGE2, aggrecanases, and MMPS. The form of glucosamine appears to influence its activity, with GHCl and GS appearing to inhibit cartilage degradation more consistently than NAG in vitro. In LPS stimulated equine explants, GS (3.075mg/ml) inhibited NO production, and PG release at 30.75mg/ml, consistent with previous findings for equimolar concentrations of GHCl (Fenton et al 2000a,b). Although PG synthesis was also decreased, tissue PG content was significantly higher. A higher dose was required to inhibit PG release in rhIL-l stimulated explants compared to LPS stimulated explants. N-acetyl-glucosamine had more variable effects, and generally did not inhibit NO production or PG release at any concentration. However, in another study, high concentrations of NAG were shown to be effective in suppressing IL-l induced COX 2 and iNOS expression (Shikhman et a1 2001). Both GS 47 and GHCl contain a free amine group on the second carbon atom, the presence of which may be important for activity of these compounds. Glucosamine inhibits NO and PGE2 production in equine cartilage stimulated with LPS, rhIL-lB and reIL-10 (Fenton et al 2000a,b; Orth et a1 2002). In general, higher doses were required to inhibit NO production (lmg/ml) compared to PGE2 production (0.5mg/ml) (Orth et al 2002). Glucosamine (4.5g/l) also decreased PGE2 both in the presence and absence of IL-1 in rat chondrocytes (Gouze et al 2001). In human OA chondrocytes, GS (1.0g/L), but not NAG, inhibited COX 2 gene expression and protein synthesis and inhibited PGE2 synthesis induced by IL-1 (Largo et al 2003). A potential benefit of glucosamine over NSAIDS was also demonstrated in this study, with glucosamine having no effect on COX-1 production. Glucosamine hydrochloride (100 ug/ml) inhibited PGE2 production but not COX 2 mRNA expression in IL-1 stimulated normal and 0A chondrocytes, and suppressed PGE2 production in synoviocytes (500 ug/ml); however, this effect was not statistically significant (Nakamura et a1 2004). NO production was suppressed in normal chondrocytes but not OA chondroyctes or synoviocytes. Glucosamine dose dependently inhibits aggrecanase-mediated cleavage of aggrecan in bovine cartilage (Sandy et al 1998, Patwari et al 2000). Mannosarnine, a structural isomer of glucosamine, also inhibited aggrecan cleavage and, along with glucosamine, was shown to inhibit IL-l induced alterations in mechanical properties of cartilage (Patwari et al 2000). Long-term exposure to glucosamine and mannosarnine inhibits aggrecanase- 48 mediated degradation of aggrecan in a dose dependent manner (up to lOmM) in retinoic acid stimulated bovine cartilage (Ilic et a1 2003). The effect on aggrecanase activity has been documented in both IL-1 and retinoic acid stimulated cartilage, suggesting that glucosamine may have a site of action downstream of IL-1 signaling pathways; however, the precise mechanism by which glucosamine inhibits aggrecanase activity requires further elucidation. Effects on MMP gene expression, protein synthesis and activity have been investigated in a number of studies. In LPS stimulated equine explants, gelatinase and collagenase activity was inhibited by GHCI at 0.25mg/ml (Fenton et al 2000b), with higher concentrations (2.5mg/ml) required to exhibit the same effect in IL-1 stimulated explants (Fenton et al 2002). However, stromelysin activity has been inhibited with a lower concentration (0.25mg/ml) in reIL-10 stimulated equine explants. Glucosamine hydrochoride had negligible effect on preformed MMP, but did inhibit both MMP protein and mRNA expression (Byron et al 2003). MMP l3 expression was inhibited to a greater degree by glucosamine than was MMP 1 and 3 activity (1.5mM-50mM and 3mM — 50mM respectively). Similar results were found in terms of mRNA expression; however, this was not a statistically Significant difference. Glucosamine decreased MMP 9 but not MMP 2 activity, and inhibited MMP 13 production (Orth et al 2002). MMP 3 production and activity has been dose dependently inhibited by GS (1.0-150uM) in human OA chondrocytes (Dodge et al 2003); however, chondrocytes obtained from 40% of OA patients did not respond. Further, no effect on MMP 1 activity was demonstrated. Reduction in MMP 3 mRN A expression has also been documented in rat chondrocytes in 49 vitro (Gouze et al 2001). GHCl suppressed MMP 1, 3 and 13 activities in IL-1 stimulated normal human chondrocytes (100 ug/ml) and synoviocytes (100 ug/ml), but not OA chondrocytes (N akamura et al 2004). High doses of glucosamine may have a detrimental effect on chondrocyte viability in vitro. GAG concentration and cell viability were reduced in canine chondrocytes cultured in alignate in the presence of glucosamine (0.1mg/ml for 12 days) (Anderson et al 1999), conversely, no detrimental effect on chondrocyte metabolism was observed with long- term exposure to glucosamine at 1 mg/ml (Ilic et a1 2003). In bovine cartilage explants, GHCI (2.5-25 mg/ml) induced a dose dependent decrease in PG synthesis and lactate production, with cell viability reduced by over 90% at the higher dose (de Mattei et al 2002). Similarly, cell viability was reduced by lOmg/ml GHCl in bovine cartilage explants in another study (Mello et al 2004). This suggests that the protective action against cytokine induced catabolic effects observed at higher doses in previous studies were due to unrecognized toxic effects (Fenton et al 2000a,b). Other potential actions in chondrocytes include reduction of phospholipase A2 and increased production of protein kinase C (Pipemo et al 2000). Glucosamine (0.1-lmM) suppressed neutrophil function and immune activity in synovial tissue, including suppression of superoxide anion generation, inhibition of enzyme release from granules and inhibition of phagocytosis (Hua et al 2002). Suppression of T cell activation has also been documented (Ma et a1 2002). 50 Chondroitin sulfate Documented actions of CS include contribution to the pool of GAG, inhibition of degradative enzyme synthesis and stimulation of GAG and collagen synthesis. A stimulatory effect on HA has been documented in synovial membrane in vitro (Nishikawa et al 1985), and CS improved synovial fluid viscosity by increasing HA concentration in human patients with knee OA (Conte el al 1991, Ronca et al 1998). Stimulation of PG production was documented in human chondrocytes in clusters, with no effect on either basal DNA activity or PGE2 production (Bassleer et al 1992). Chondroitin sulfate was more active in counteracting IL-l induced effects on PGE2, PG (500—1000ug/ml) and type II collagen (100-1000ug/ml) during the period of cluster formation (day 0-16) than in the period after which clusters had formed (PG: 100- lOOOug/ml, type II collagen and PGE2 lOOOug/ml) (Bassleer et a1 1998). In a rabbit model of OA, cartilage PG content was significantly higher following administration of CS prior to induction of OA (Uebelhart et a1 1998). In equine explants, CS decreased NO production, PG degradation and MMP 13 production, albeit at different concentrations (0.25mg/ml, 0.5mg/ml, and 0.125mg/ml respectively) (Orth et al 2002). Chondroitin sulfate had no effect on PGE2 production, and had a minimal not statistically significant effect on MMP 2 and 9. Collagenase, phospholipase A2, and n-acetylglucosaminidase activity were decreased in synovial fluid obtained from human patients with knee OA treated with CS (Ronca et a1 1998). A protective effect in terms of PG concentration was recently established using human 51 chondrocyte cultures under conditions of IL-1 stimulation and cyclic pressurization (Nerucci et al 2000), suggesting that CS may be more effective when chondrocytes are exposed to mechanical forces. Chondroitin sulfate (100 ug/ml) may also protect chondrocytes from N0 mediated apoptosis following stimulation with IL-1 (Conrozier 1993). Other potential mechanisms of action have been postulated. Chondroitin sulfate partially inhibits complement (Paroli et al 1991) and elastase activity in human leukocytes (Baici et a1 1994). Leukocyte elastase activityiss inhibited more efficiently by low molecular weight CS compared to intact CS (Cho et al 2004). In a collagen-induced arthritis model, C-4-S significantly reduced the production of free radicals and restored endogenous antioxidant levels (Campo et al 2003a, b). Further, CS inhibits intra-articular bradykinin induced PG depletion in rats in a dose dependent manner (Omata et al 1999). Glucosamine and chondroitin sulfate in combination The combination of glucosamine and CS appears to be more effective than either product alone. In equine explants, GHCI and CS in combination decreased LPS induced NO production (0.5mg/ml and 0.25mg/ml), and inhibited MMP 13 production (0.5mg/ml and 0.125mg/ml) (Orth el al 2002). The same dose of glucosamine had minimal or no effect alone. The combination decreased PG degradation (lmg/ml and 0.25mg/ml), with a higher dose of CS alone required to exhibit a similar effect. In bovine articular cartilage in vitro, Cosequin® (GHCI, LMWCS, manganese ascorbate) increased PG synthesis and 52 enhanced a mild stimulatory effect of a NSAID on PG synthesis, as well as inhibiting the NSAID induced acceleration in ECM degradation (Lippiello et a1 2002). In a canine cranial cruciate ligament model of OA, the same product reduced cartilage degradation and increased CS epitopes (3D3 and 7D4) and GAGS in synovial fluid (Johnson et al 2001). In a rabbit instability model of OA, severity of cartilage lesions in terms of both area and severity was significantly reduced (Lippiello et al 2000). The combination had a synergistic effect, with a greater effect than either compound alone. In vivo joint stress was simulated in vitro by matrix depletion with enzymes, heat stress, and mechanical compression (Lippiello 2003). Exposure of cartilage explants to pronase induced upregulation of PG synthesis, whereas hyaluronidase, chondroitin ABC lyase and stromelysin had an inhibitory effect. Cosamin® (GHCI, LMWCS and manganese ascorbate) supplementation had no influence on the inhibitory effect of hyaluronidase and chondroitin ABC lyase. PG synthesis was stimulated in pronase treated aged joints with high doses (400ug/ml), while a lower dose (lOOug/ml) reversed inhibition of PG synthesis induced by stromelysin. Heat stress similarly decreased PG synthesis, an effect prevented by administratin of GHCl and CS in combination. Cartilage from aged animals was more responsive to stress in terms of PG synthesis and also to supplementation with Cosamin®, with a 1000% increase in PG synthesis. 53 Effect on IL-1 signaling pathways Recently, the effects of glucosamine on IL-1 signaling pathways have been investigated. Using human OA cartilage, the effects of GS, galactosamine hydrochloride and NAG on AP-l and NFKB were investigated (Largo et al 2003). Glucosamine sulfate alone did not modify NFKB binding; however, significantly inhibited NFKB binding in a dose dependent manner in IL-1 stimulated cartilage, with maximal inhibition at 1000mg/L. Further, GS prevented IL-l induced translocation of p50 and p65 subunits of NFKB to the nucleus, and prevented IL-l induced degradation of IKB. No effect on AP-l binding was observed, consistent with findings using rat chondrocytes where glucosamine (4.5gm/L) inhibited NFKB but not AP-l pathways (Gouze et a1 2002). Galactosamine hydrochloride and NAG had no effect in human OA cartilage (Largo et al 2003), in support of other studies where NAG had no effect on NFKB translocation or JNK, ERK or p38 activation (Shikhman et al 2001). AS NFKB induces transcription of COX 2 and iNOS, the anti- inflammatory effects of glucosamine on PGE2 and NO production observed in these studies (Gouze et a1 2002) may be due to inhibition of NFKB binding to its response elements. Other potential beneficial influences of glucosamine on IL-1 signaling have been documented at the level of IL-1 receptors. Glucosamine increased expression of IL-lRII (Gouze et al 2002). IL-IRII is a truncated protein and functions as a decoy receptor, by effectively trapping IL-1, the inflammatory effect of which requires signaling through IL- lRl. There was no effect on IL-lRa mRNA expression. 54 In other cell types such as mesangial cells and adipocytes, glucosamine modulation of gene expression involves the transcription factor Specificity Protein (Spl) (Goldberg et al 2000). This transcription factor has binding sites in promoters of genes coding for MMPS and aggrecan (Valhmu et al 1995); however, the regulation of Sp transcription factors in cartilage has not been determined. Whether CS is capable of exerting a Similar effect on IL-1 signaling requires further investigation. Pharmacokinetics Glucosamine Glucosamine is a small water-soluble molecule (molecular weight 179), with a pKa that favors its intestinal absorption and intracellular transportation (Kelly 1998). Absorption is carrier-mediated, whereas absorption of NAG occurs via diffusion (Tesoriere et a1 1972). Quantitative aspects of glucosamine absorption have been debated. Early pharmacokinetic and bioavailability studies utilized colorimetric, chemical or radiolabeling techniques; however, limitations were apparent with each analytical method. Colorimetric assays failed to differentiate between glucosamine and other hexosamines, and required the administration of doses far exceeding levels considered therapeutic and/or achievable in animals (Setnikar et al 2001). Following intravenous (IV) and oral (PO) administration of crystalline GS in man, plasma and urinary concentrations were assayed using plasma deproteinised with sulfosalicylic acid or ion exchange chromatography (Setnikar et al 1986). Oral GS at four times the recommended daily dose, resulted in plasma levels were still below the limit of detection (3 jig/ml) with 55 ion exchange chromatography. Thus the ADME profile (absorption, distribution, metabolism and excretion) of glucosamine required further elucidation. Limitations with radiolabeling techniques were also evident. Accurate determination of bioavailability of glucosamine was precluded as both the labeled compound and its metabolites were quantified. However, such techniques did enable preliminary investigation of pharmacokinetics in a number of species. Incorporation into plasma proteins, biotransfomation in the liver and urinary excretion was demonstrated following IV closing in a rat (12.6mg/kg) (Setnikar et al 1984). Tissue distribution was rapid, with early incorporation into articular cartilage (confirmed by autoradiographs of sagittal sections of necropsied animals). Most organs, including articular cartilage, contained some radioactivity even 144 hours after administration. The equivalent dose administered orally resulted in peak plasma concentration by 4 hours, 95% absorption and an estimated bioavailability of 40% based on urinary excretion within the first Six hours. Again, early incorporation into articular cartilage was observed (within 30 minutes), and concentrations from 8 hours were similar to that in plasma. Another study in the rat utilizing oral doses at 4.6, 46, 125 times the recommended daily dose, demonstrated that absorption and elimination kinetics were independent of the dose, however at all sampling times radioactivity content was dose related. Significantly higher levels of radioactivity existed in cartilage compared to plasma at 72 and 120 hours, suggesting substantial incorporation of glucosamine into articular cartilage. Multiple dosing (SID PO 12.6mg/kg for 6 days) lead to accumulation of glucosamine in 56 cartilage, with 3-5 times greater radioactivity at 144 hours after the last of the multiple doses compared to after a single dose. Furthermore, at the same time period, cartilage radioactivity was nearly 3 times greater than in plasma after both single and multiple dose administration (Setnikar et al 2001). Radiolabeling studies in man have demonstrated similar pharmacokinetics (Setnikar et al 1986 and 1993). Nearly 90% absorption was noted following oral administration, and, whilst no free glucosamine was detectable in plasma (below LOQ), the incorporation of radioactivity into plasma proteins was found to follow Similar pharmacokinetic patterns as parenteral administration, albeit at a concentration approximately 5 times lower. A Similar ADME profile occurs in dogs. Two hours following IV administration (12.6mg/kg), radioactivity in articular cartilage was 13 times deproteinised plasma levels and twice plasma protein concentrations (Setnikar er al 1986). Tropism for articular cartilage was again apparent with radioactivity still detectable 144 hours after PO administration. Conversion of GS into radiolabelled galactosarrrine moieties of CS and keratan sulfate in cartilage was also demonstrated (Dodge et al 2001). High Performance Liquid Chromatography (HPLC) allows for accurate determination of glucosamine in orally available products and pharmacokinetic studies. As glucosamine lacks a chromophore absorbing in the ultraviolet light range, modifications were required. Pre-column derivatization of GHCl with phenylisothiocyanate overcame this problem and was shown to be specific (thereby eliminating interference by degradation products), 57 accurate and precise. Plasma concentrations were measured between 1.25 — 20 ug/ml in dogs (Liang et al 1999). Use of a plasma concentration versus time curve in a dog (2000 mg GHCl PO) established a maximum plasma concentration at less than 2 hours, with none detectable at 8 hours. Due to interference from similarly derivatized endogenous amines, this technique was not applicable to studies in the rat, prompting development of a technique using HPLC with pre-column derivatization and ion exchange purification. The LOQ was found to be 1.25 ug/ml, sufficient for pharmacokinetic studies with 350mg/kg PO; however, lower doses could not be determined (Aghazadeh-Habashi et al 2002b). Rapid elimination and distribution of glucosamine was also apparent (Aghazadeh-Habashi et al 2002a). Intraperitoneal dosing exhibited complete bioavailability, with poor oral bioavailability of glucosamine thought due to loss in the gastrointestinal tract rather than hepatic first pass effect as previously thought (Setnikar et a1 1993). Other analytical methods, such as refractive index detection, have also been utilized and found to be accurate, precise and useful for the quantification of GS, although with less accuracy than HPLC (El-Saharty et 012002) More sensitive and accurate techniques that eliminate interference by degradation products have enabled the determination of plasma concentrations achievable after oral administration in the dog and horse (Adebowale et al 2002, Du et al 2004). Results of single dose pharmacokinetics of glucosamine using these methods support its absorption, with lower oral bioavailability in the horse than in the dog. In the dog, glucosamine was 58 rapidly absorbed with a Cmax between 7.1-12.1 jig/ml. In the horse, after IV administration (Cmax 349 ug/ml), concentrations decreased rapidly in a biphasic manner, and a high volume of distribution was observed, attributed to the extensive uptake of glucosamine into tissues. In the horse, oral doses at currently recommended rates resulted in plasma levels that were below the limits of quantification, necessitating the use of doses approximately 5-10 times greater. Administration of 125mg/kg PO resulted in a Cmax of 10.6 rig/ml. Recently, a single dose of 20mg/kg GHCl was admininstered to horses both IV and via nasogastric intubation (Laverty et al 2005). AS expected, mean maximum serum concentrations were greater following IV compared to PO administration (288 +/- 53 uM and 5.8 +/- 1.7uM respectively). Synovial fluid levels were also measured l-hour later at 9-15 uM and 0.3-0.7 uM for IV and PO administration; however, glucosamine was still detectable in synovial fluid from most horses 12 hours after dosing. Chondroitin sulfate Similar analytical methods have also been used for the quantification of CS following oral administration. Early studies of intact CS utilized capillary electrophoresis (Pervin et a1 1994, Volpi 1996) or fluorometric labeling followed by HPLC on hydroxyapatite (Narita et al 1995) or size-exclusion columns. The latter technique has been used to quantitatively measure CS both in raw materials and dosage forms (Choi et al 2003), and also to determine plasma concentrations (Conte et a1 1995). Limitations, such as lack of sensitivity to distinguish between CS disaccharides, were apparent in early studies. The 59 methodology used appeared to influence levels of CS that were measured, with HPLC more accurate than spectrophotometric methods for quantification of CS in dosage forms (Choi et a1 2003). This prompted the development of other analytical methods incorporating enzymatic digestion to detect the disaccharides, with applications including their detection in plasma (Huang et al 1995, Kinoshita et al 1999) and urine (Sakai et al 2002) Early studies did not support the oral absorption of CS, as evidenced by the lack of change in serum GAG levels when measured with dimethylene blue (Baici et al 1992). Such studies have since been criticized due to the low sensitivity inherent with colorimetric methods, and as dimethylene blue determines all uncharged GAGS not just CS (Lualdi et al 1993). Other studies attributed lack of absorption of CS following oral administration to an inability to cross the gastrointestinal tract due to its high molecular weight (Anderrnann et al 1982). The absorption of CS was evaluated using CaCO2 cell monolayers, an in vitro model of intestinal epithelium (Cho et al 2004). LMWCS traversed the membrane better than a higher molecular weight CS. Using a spectrophotometric assay, differential absorption of CS along the intestinal tract of rats was depicted (Barthe et a1 2004). CS was transported across the small intestine in its intact form, probably by endocytosis. A greater amount was transported in the large intestine in the form of its constituent disaccharides, with degradation in the colon and cecum. 60 Similar to glucosamine, radiolabeling techniques have been used to investigate the metabolic fate of CS. In rats and humans, oral CS was rapidly absorbed and tropism for articular cartilage was demonstrated with scintigraphy (Ronca et a1 1998). In the rat and dog, oral absorption of a single dose of 50% C—4-S and 50% C-6-S was greater than 70%, with plasma radioactivity levels increasing rapidly (Palmierei et al 1990). In the rat, distribution of radioactivity in tissues was the same regardless of the route of administration (IM and PO). Chondroitin sulfate rapidly accumulated in cartilage, with cartilage radioactivity levels at 72 hours exceeding levels in other tissues including plasma. Cartilage levels were greater with oral rather than IM administration in the rat. In the dog, synovial fluid concentrations were also measured (depicted as total radioactivity/ml independent of the nature of radioactive compounds which may have been present). Five hours after oral dosing, radioactivity was 66.5% higher in synovial fluid than plasma (10.1 ug/ml and 6.0 ug/ml respectively). In plasma and synovial fluid, molecular weights higher than that administered were observed, presumably due to protein binding. Chromatography of rat articular cartilage at 24 hours after oral administration yielded both high and low molecular weight compounds, indicative of the metabolism of chondroitin sulfate. Both radiolabeling techniques and size exclusion chromatography were combined to investigate pharmacokinetic properties in the rat and dog (Conte et a1 1991 and 1995), and supported more than 70% absorption found in other studies (Palmieri et al 1990). Gel filtration of plasma and synovial fluid of dogs at 3 and 5 hours post administration depicted the presence of both native labeled compound and N-acetyl-galactosamine, with 61 radioactivity 66.5% higher than in plasma. Radioactivity accumulated in both synovial fluid and articular cartilage in rats, with mostly high molecular mass CS present in cartilage at 24 hours. Both synovial fluid and cartilage radioactivity levels exceeded plasma levels at 24 hours, with accumulation in cartilage depicted by greater radioactivity at 48 compared to 24 hours. Radioactivity in cartilage exceeded radioactivity in other all other tissues by 48 hours. Pharmacokinetics have been investigated in humans with a single dose of 0.8g CS once daily or in divided doses twice daily (Conte et al 1995). Both dosage regimes resulted in a significant increase in plasma concentration, with no significant difference in time to peak concentration noted. However peak plasma concentration (Cmax) was higher with SID dosing (2.6 rig/ml SID and 1.2 ug/ml BID). Plasma levels were almost constant (around 1.8 ug/ml), with a plateau reached in 2-3 days with SID dosing throughout a 30- day trial conducted in patients with first and second degree OA (based on American Rheumatism Association designation). Oral bioavailability and pharmacokinetic parameters were investigated in man using a single (4g) oral dose of CS of bovine origin (Condrosurf®) and shark origin (Volpi 2002 and 2003). With the bovine CS, plasma levels CS increased more than 200% from endogenous levels. Basal endogenous disaccharide composition of plasma was approximately 60% non sulfated disaccharide and 40% 4-sulfated disaccharide, with administration of CS decreasing relative amount of non sulfated disaccharide and increasing both 4- and 6-Sulfated disaccharide. Maximum plasma concentration was 62 12.73 ug/ml, higher than that obtained with CS from shark cartilage (Cmax 4.9 rig/ml). Absorption was also slower. A recent study utilizing pre-column derivatization, HPLC and fluorometric detection showed good resolution of CS disaccharides, with a level of sensitivity of 20-3011g (V olpi 2000). This technique was recently adapted for the detection of CS disaccharides in dog and horse plasma, and was shown to be valid and specific with high recovery (limit of detection of lug/ml) (Du et al 2002). An integral component of this study was the investigation of the pharmacokinetics of CS following intravenous administration in dogs (400mg) and horses (3 g). Plasma disaccharide concentrations of 350 ug/ml (dog) and 110 pg/ml (horse) were rapidly achieved, with concentrations below 20 ug/ml by 20 hours post administration in the horse. Oral absorption (3g) has been demonstrated in the horse, with a Cmax of 36.5 jig/ml (Eddington et a1 2001). Absorption was rapid (tmax 1.32h), with an apparent bioavailability of 22%. Different forms of CS were compared, with the 16.9kDa forms, the active ingredient in Cosequin®, having statistically higher plasma concentration and area under the plasma concentration curve (AUC) compared to an 8kDa product. Glucosamine and Chondroitin sulfate in combination The bioavailability and pharmacokinetics of GHCI and LMWCS in combination have been investigated in both the horse and dog (Du et al 2004, Adebowale et al 2002). Plasma concentrations of glucosamine (Liang et al 1999) and chondroitin sulfate (Du et al 2002) were quantified using techniques previously described. Single dose 63 pharmacokinetics was investigated in the horse, with a Cmax of 349 ug/ml and 10.6 ug/ml following IV and PO administration for glucosamine and 210 ug/ml and 36.5 rig/ml for CS. In the dog, single oral doses of 1500mg GHCl and 1200mg CS, or 2000mg and l600mg were administered, and multiple dose pharmacokinetics investigated with BID doses of 1500mg GHCl and 1200mg CS from days 1-7 then 3000mg GHCl and 2400mg CS on days 8-14. The mean bioavailability of glucosamine after single and multiple dosing (12.1-12.7% and 9.7-10.6%) was lower than that previously estimated in radiolabel studies. Radiolabeling may overestimate systemic bioavailability due to failure to detect presystemic metabolism in gastrointestinal tract or liver during absorption as drug and metabolites are not differentiated. There was no significant difference found between single and multiple dose pharmacokinetics. Higher levels of constituent disaccharides ADi-4S and ADi-6S were found compared to ADi-OS, correlating to the low molecular weight chondroitin used in the study (Cosequin® - contains approximately 60% C-4-S and 40% C-6-S). The absorption of glucosamine and CS were rapid with Cmax of 8.95- 124 ug/ml and 19.0-21.5 jig/ml respectively (tmlx around 1.5h). Unlike glucosamine, CS disaccharides accumulated in plasma after multiple dosing depicting a significant carry over effect. Cmax was 96.3 and 208 ug/ml with P0 1200mg on day 7 and 2400mg on day 14 respectively, with 200% and 278% bioavailability of total disaccharides on a molar basis after multiple dosing with 1200mg and 2400mg respectively. 64 Clinical Trials Human A number of clinical trials have been performed in man with mostly favorable, albeit equivocal, results. Flaws in terms of study design (low number of participants, lack of placebos, inclusion criteria) and assessment of outcome (subjective) were evident in early studies. However moderate symptomatic effects and a good safety profile were demonstrated in a number of short-term trials (Busci et al 1998, Leffler er al 1999, Muller-Fassbender et al 1994, Noack et al 1994). Recently, a meta—analysis and quality assessment of 15 randomized double blind placebo controlled studies in patients with hip or knee 0A was reported (McAlindon et al 2000). Of these trials, all but one showed positive results in terms of decreasing pain and improving mobility, with overall moderate effects for glucosamine and large effects for chondroitin. However, deficiencies were noted in terms of randomization, blinding and completion rates. Furthermore, publication bias was suggested with most of the trials supported by manufacturers’ of these products. In two randomized double blind placebo controlled trials in patients with knee OA conducted over a six-month period, no additional beneficial analgesic effects of glucosamine were seen compared to placebo (Hughes et a1 2002, Rindone et al 2000); however, a statistically significant difference in degree of knee flexion was noted in the first study. Symptoms were scored based on both subjective assessment and recommended grading scales (visual analog and WOMAC score (Western Ontario and 65 McMaster University CA index)). Of note, in both studies patients generally had more severe OA than those in other trials. Two recent long-term randomized double blind placebo controlled trials have identified a beneficial effect of glucosamine sulfate (1500mg/day) in knee OA (Reginster et a1 2001, Pavelka et a1 2002). Patients had mild to moderate OA based on American College of Rheumatology classification criteria (Altman et a1 1986). In the first study, glucosamine resulted in a 20-25% improvement with respect to pain and physical function, with worsening WOMAC scores evident in placebo treated patients. Placebo patients also had progressive joint space narrowing (measured as mean joint Space width of the medial compartment of the tibiofemoral joint and minimum joint space width), while glucosamine patients had no significant joint Space loss. No significant adverse effects were seen, and no alteration in glycemic homeostasis or other routine laboratory tests were identified. Based on analysis of mean joint space width in the same population, patients with mild OA appeared to be the most responsive to supplementation with glucosamine (Bruyere et al 2003). Similar results in terms of symptomatic improvement, slowing of radiographic progression and lack of perceivable joint space narrowing were found in the second long-term study. Since these trials have been published, results have been criticized. These trials utilized a standing antero-posterior fully extended knee view, the recommended “gold standard” (Altman et al 1987). It was postulated that by providing symptomatic relief, knee positioning might have been altered, thereby introducing potential systematic error in using measurement of joint space width as an index of OA progression. However, further analysis revealed that pain was not a 66 confounder in joint space narrowing assessment on this radiographic view (Pavelka et al 2003). Further, patients with high cartilage turnover, as measured by urinary collagen type II C-telopeptide fragments, appeared to be most responsive to supplementation with glucosamine (Christgau et al 2004) Fewer trials have been conducted to determine the efficacy of chondroitin sulfate. In a one-year randomized double blind placebo controlled study of knee OA (Uebelhart et al 1998), CS significantly reduced pain and increased overall mobility. In addition, joint Space narrowing was reduced and biochemical markers stabilized. Results of a similar trial conducted over 6 months were also supportive (Busci et al 1998), with CS well tolerated in both trials. In a 3-month trial, CS resulted in Significant improvement in clinical symptoms, including joint mobility, with no difference in efficacy appreciated between different dosage regimes (SID versus divided TID) (Bourgeois et al 1998). In a 3-year randomized double blind placebo-controlled trial, CS was found to be protective against radiographic progression of finger joint DA (V erbruggen et a1 1998). Recently, intermittent administration of CS (3 months twice over 1 year) was also found to reduce pain and joint space narrowing in patients with knee OA, suggestive of a prolonged chondroprotective effect (Uebelhart et a1 2004). In a placebo-controlled study of patients with knee OA, synovial fluid parameters improved following CS administration of 3 grams orally once daily. After 5 days, there was a significant decrease in the lysosomal enzyme N-acetylglucosaminidase, and no significant difference in leukocyte count and protein levels. HA levels increased 67 concurrent with an alteration in molecular mass distribution, resulting in an increase in high molecular weight fractions. The molecular mass of sulfated GAGS also changed. High molecular weight molecules, indicators of cartilage breakdown, decreased and low molecular weight molecules increased, suggestive of the incorporation of exogenous CS (Conte et al 1991). Such changes in synovial fluid parameters were supported by another study (Ronca et al 1998). Trials utilizing both glucosamine and chondroitin sulfate in combination have been limited, and have used commercially available products that also contain manganese ascorbate. In a randomized placebo controlled study (Cosamin DS®- GHCl 1000mg, CS 800mg and manganese ascorbate), patients with radiographically mild to moderate OA had significant improvement at 4 and 6 months, whereas there was no significant improvement in patients with radiographically severe OA based on the Lesquene Index of severity (Das et al 2000). In a 16 week randomized double blind placebo controlled study of patients with knee or lower back DJD, therapy relieved symptoms of knee OA (based on patient assessment, visual analog scale and physical examination score), but not spinal DJD (Leffler et al 1999). In a meta-analysis of oral glucosamine and CS in knee OA, structural efficiacy of glucosamine and sympotrnoatic efficacy of both products were demonstrated. This study included analysis of a number of the studies described previously (Richy et al 2003). Recently the efficacy of glucosamine has been compared to other commonly used OA drugs. Glucosamine was found to have greater benefits in terms of reduction of pain 68 compared to a NSAID (ibuprofen) in a short-term trial of patients with temporomandibular joint OA (Thie et a1 2001). In another study comparing CS and a NSAID, although NSAID treated patients had prompt reduction of clinical symptoms, signs reappeared following cessation of treatment. Of interest, while the therapeutic response to CS appeared later, benefits lasted up to 3 months after cessation of treatment (Morreale et al 1996). The safety profile of glucosamine and chondroitin has been supported by a number of clinical trials, including those reported above (Towheed et al 2001). Initially glucosamine was thought to be associated with the development of diabetes; however, continuous intravenous infusion was required to induce insulin resistance (Patti er al 1999, Monauni et al 2000). In a recent placebo-controlled study with short-tenn intravenous infusion of glucosamine, no effect on insulin-induced glucosamine uptake was evident (Pouwels et al 2001). Further, results of a recent placebo controlled double blind randomized clinical trial involving patients with Type 2 diabetes, showed that oral glucosamine and CS supplementation had no clinically significant effect on glucose metabolism (Scroggie et al 2003). Reported side effects are rare with angioedema reported in one patient due to an immediate hypersensitivity reaction to GS (Matheu et al 1999). Small Animal In recent years, a number of trials have been performed in small animals, predominantly in the dog. The majority of these trials have utilized GHCl and LMWCS in combination (Cosequin®). In a rabbit instability model of OA, the combination had a 69 chondroprotective effect, with absence of severe lesions (Mankin grade 7 or greater) along with significant reduction in total linear involvement and total grade histologically. (Lipiello et a1 2000) This effect was greater than that seen for either agent alone. In a double blind placebo controlled study, dogs were administered Cosequin® or placebo for 21 days prior to the induction of short-terrn synovitis in the carpus with infra-articular chymopapain (Canapp et al 1999). Preadministration Significantly reduced both soft tissue phase scintigraphic uptake at 48 days and bone uptake at 41 and 48 days, concurrent with a reduction in the degree of lameness. Due to the structural similarity of GAGS and heparin, the concurrent use of these products with other platelet inhibitors, such as phenylbutazone or aspirin, is often cautioned (Davidson 2000, Malone 2002). A good safety profile has been demonstrated in the cat and dog (McNamara er al 1996 and 2000) following oral administration of Cosequin® at doses exceeding the recommended daily dose for 30 days. Biochemical, hematological and hemostatic indices remained within normal limits, with only minor but not clinically significant changes in hemoglobin content and total white cell count. There was no effect on clotting times. In a study in rats, the therapeutic margin with regard to prolonged administration was more than 10-30 times more favorable for GS than for the NSAID indomethacin (Setnikar et al 1991). In a double blind placebo controlled study of oral Cosequin® in a cranial cruciate rupture model of 0A in dogs, treated dogs had decreased mean modified Mankin scores (Hulse et al 1998). Using the same model of OA, long-term (5 month) administration of the same 70 product resulted in modulation of articular cartilage metabolism reflected by an increase in synovial fluid 383 and 7D4 epitope concentrations (Johnson et al 2001) - chain length and sulfation patterns of chondroitin sulfate alter early in OA, exposing unique epitopes that can be detected by immunoassay with monoclonal antibodies (Hardingham 1998). Systemic effects were also noted with similar changes in the contra-lateral non-operated joint. There was however a delay of four weeks before GAGS with 383 epitope were released into the synovial fluid. A 42% increase in serum GAGS was noted in dogs in a 30-day trial using the same product (Lippiello et al 1999). Further, when this serum was used to incubate cartilage segments in vitro, GAG biosynthetic rate significantly increased by 50%, with a concomitant reduction in proteolytic degradation of 59%. Recently, a multi-centered, double blind, randomized clinical trial was initiated to compare a glucosamine/chondroitin sulfate preparation (containing C-4-S, GHCl, NAG, ascorbic acid and zinc sulfate) to carprofen, a NSAID (McCarthy et al 2003). Dogs included in the study had radiographic evidence of hip or elbow 0A and were treated for 70 days, with reassessment one month following cessation of treatment. Preliminary results were encouraging with improvement in clinical parameters such as lameness. Equine To date, clinical trials in the horse have been limited. A randomized double blind placebo controlled clinical trial was conducted in horses 5-15 years of age with progressive forelimb lameness of 3-12 months duration due to navicular disease. Horses were administered Cosequin® (9gms GHCl, 3gms LMWCS, 600mg manganese) or placebo 71 twice daily for 56 days. A statistically Significant improvement in overall clinical score, overall clinical condition and total lameness was observed, with no side effects reported (Hanson et a1 2001). Another study into the safety profile of this oral dosage form was conducted in which horses were administered 5 times the recommended daily dose for 34 days (Kirker-Head et al 2001). No clinically significant changes in hematological, biochemical or synovial fluid values were observed, and no adverse effects noted. Of interest, hematocrit, hemoglobin levels, and total white cell count were significantly increased. Cosequin® was also used in a 6 week study of possible beneficial effects in the treatment of 0A in 6-20 year old horses (Hanson et al 1997). Physical examination, intra-articular analgesia, radiographs and/or fluoroscopy were used to confirm a diagnosis of OA of the distal interphalangeal, metacarpophalangeal, tarsometatarsal or carpal joints. Within two weeks, a significant improvement was noted in lameness grade, flexion test grade and stride length. By four weeks, there was no further improvement in lameness grade and no significant changes in other parameters. Age was not found to be a significant factor. A number of problems with trial design Should be recognized, including lack of placebos and lack of blinding of assessors to treatment method. Furthermore, as most horses were able to return to exercise and competition after the initial two wks of treatment, this may explain the leveling in improvement seen after 2 weeks. Again, no side effects were noticed. 72 Other studies using Cosequin® have had conflicting results. Lameness measured by force plate analysis resolved (Hanson 1996); however, no beneficial effects were seen in a Freunds adjuvant model of synovitis (White et al 1994). The efficacy of IM NAG and CS was found to be significantly less than that of a PSGAG using the same model (White et a1 2003). Recently a double blind placebo controlled study was performed using precursors of glucosamine (Cortaflex®: containing glutamine, glutamic acid, glycine and glucuronic acid-loading dose 60mlS/day 5 days then 30mls per day) (Clayton et al 2002). Horses included in the study had OA of the distal intertarsal and/or tarsometatarsal joints. Using force plate analysis, an improvement in gait symmetry measured by vertical ground reaction force was evident. In two studies of the effect of supplementation with glucosamine on serum markers, no significant influence on keratan sulfate, osteocalcin or pyridinoline crosslinks were observed (Fenton et al 1999, Caron et a1 2002). Horses in these studies were clinically normal with no evidence of CA. The efficacy of exogenous intramuscular and oral chondroitin sulfate in an experimentally induced model of equine arthritis has been investigated (Doma et al 1998). Both routes of administration were associated with improved joint function and reduced lameness scores. Time of onset for clinical improvement was Slower with oral administration however the maximum level of improvement achieved was comparable for both routes of administration. 73 Cell Membrane IL-l OOOOOOOOOOOOOOC QCQQQJQQOQQDOQQQQO {:1 I?" OOOOOOOOOOOOOO" E g SOOOOOOOOOJOOOCOOO “U f‘ Z ‘< U co co 3. '5 5‘; 7‘ -o ( ECSIT 2 6 / \* TRAF6 E TAB] 2 _ TABZ j NIK AP-l binding site NFKB binding site Figure 1 (chapter 1): Interleukin-1 Signaling Pathways. 74 References Adebowale A, Du J, Liang Z, et al. The bioavailability and pharmacokinetics of glucosamine hydrochloride and low molecular weight chondrotin sulfate after single and multiple doses to beagle dogs. Biopharm Drug Dispos 2002;23:217-25. Aghazadeh-Habashi A, Sattari S, J amali F, Pasutto F. Single dose Pharmacokinetics and Bioavailabilty of Glucosamine in the Rat. J Pharm Pharmaceut Sci 2002;5:181-4. Aghazadeh-Habashi A, Sattari S, Pasutto F, J amali F. High Performance Liquid Chromatographic Determination of Glucosamine in Rat Plasma. J Pharm Pharmaceut Sci 2002;5: 176-80 Aigner T, McKenna L. Molecular pathology and pathobiology of osteoarthritis cartilage. Cell Mol Life Sci 2002;59:5-18. Aigner T, Zien A, Gehritz A, et a1. Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology. Arthritis Rheum 2001;44:2777-89. Akatsuka M, Yamamoto Y, Tobetto K, et al. In vitro effects of hyaluronan on prostaglandin E2 induction by interleukin-1 in rabbit articular chondrocytes. Agents Actions 1993;38:122-5. Alaaeddine N, Di Battista JA, Pelletier JP, et al. Osteoarthritic synovial fibroblasts possess an increased level of tumor necrosis factor-receptor 55 (TNF-R55) that mediates biological activation by TNF-alpha. J Rheumatol 1997;24:1985-94. Alaaeddine N, Di Battista JA, Pelletier JP, et al. Differential effects of IL-8, LIF (pro-inflammatory) and IL-11 (anti-inflammatory) on TNF-alpha-induced PGE2 release and on signalling pathways in human OA synovial fibroblasts. Cytokine 1999;11:1020-30. Altman R, Asch E, Bloch D, et a1. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 1986;29:1039-49. 75 Altman RD, Fries JF, Bloch DA, et al. Radiographic assessment of progression in osteoarthritis. Arthritis Rheum 1987;30: 1214-25. Altman RD, Moskowitz R. Intraarticular sodium hyaluronate (Hyalgan) in the treatment of patients with osteoarthritis of the knee: a randomized clinical trial. J Rheumatol 1998; 25:2203-12. Alwan WH, Carter SD, Dixon JB, et al. Interleukin-1 like activity in synovial fluids and sera of horses with arthritis. Res Vet Sci 1991;51:72-7. Amiel D, Toyoguchi T, Kobayashi K et al. Long-term effect of sodium hyaluronate (Hyalgan®) on osteoarthritis progression in a rabbit model. Osteoarthritis Cartilage 2003;11:636-43. Amin AR, Attur M, Patel RN et al. Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage. J Clin Invest 1997;99:1231-7. Andermann G, Dietz M. The influence of the route of administration on the bioavailability of an endogenous macromolecule: chondroitin sulfate (CSA). Eur J Drug Metab Pharmacokinet l982;7:1 16 Anderson CC, Cook JL, Kreeger JM, et al. In vitro effects of glucosamine and acetylsalicylate on canine chondrocytes in three dimensional culture. Am J Vet Res 1999;60:1546-51. Andrews J L, Sutherland J, Ghosh P. Distribution and binding of glycosaminoglycan polysulfate to intervertebral disc, knee joint articular cartilage and meniscus. Arzneimittelforschung 1985 ;35 : 144-8. Apte SS. A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family. Int J Biochem Cell Biol 2004;36:981-5. Arend WP. Interleukin-1 receptor antagonist. [Review]. Adv Immunol 1993;54:167- 227. 76 Armstrong S, Lees P. Effects of carprofen (R and S enantiomers and racemate) on the production of IL-1, IL-6 and TNFor by equine chondrocytes and synoviocytes. J Vet Pharmacol Therap 2002;25: 145-53. Arner E. Aggrecanase-mediated cartilage degradation. Curr Opin Pharmacol 2002;2z322-9. Asahara H, Fujisawa K, Kobata T, et al. Direct evidence of high DNA binding activity of transcription factor AP-l in rheumatoid arthritis synovium. Arthritis Rheum 1997;40:912-8. Attur MG, Patel RN, Abramson SB, et al. Interleukin-17 up-regulation of nitric oxide production in human osteoarthritis cartilage. Arthritis Rheum 1997;40:1050-3. Auphan N, DiDonato J, Rosette C et al. Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science 1995; 270:286-90. Aupperle KR, Bennett BL, Boyle DL, et al. NF-KB Regulation by IKB Kinase in primary fibroblast-like synoviocytes. J Immunol 1999;163:427-33. Aydelotte MB, Greenhill RR, Kuettner KE. Differences between sub-populations of cultured bovine articular chondrocytes. II. Proteoglycan metabolism. Connect Tissue Res 1988;18:223-34. Aydelotte MB, Kuettner KE. Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production. Connect Tissue Res 1988;18:205-22. Baeuerle P, Baichwal V. NF-kappa B as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv Immunol 1997:65z211-90. Baici A, Bradamante P. Interaction between human leukocyte elastase and chondroitin sulfate. Chem Biol Interact 1984;51 :1-1 1. Baici A, Horler D, Moser B et al. Analysis of glycosaminoglycans inhuman serum after oral administration of chondroitin sulfate. Rheumatol Int 1992;12:81-8. 77 Bailey CJ. Wastage in the Australian Thoroughbred Racing Industry. Rural Industries Research and Development Corporation Publication No 98/52, 1998;pp1-66. Baldwin AS. The transcription factor NF-KB and human disease. J Clin Invest 2001;107:3-6. Barchowsky A, Frleta D, Vincenti MP. Integration of the NF-kappaB and mitogen- activated protein kinase/AP-l pathways at the collagenase-l promoter: divergence of IL-1 and TNF-dependent signal transduction in rabbit primary synovial fibroblasts. C ytokine 2000; 12: 1469-79. Barthe L, Woodley J, Lavit M et al. In vitro intestinal degradation and absorption of chondroitin sulfate, a glycosaminoglycan drug. Arneim-Forsch Drug Res 2004;54:286-92. Bassleer CT, Combal JP, Bougaret S, Malaise M. Effects of chondroitin sulfate and interleukin-1 beta on human articular chondrocytes cultivated in clusters. Osteoarthritis Cartilage 1998 ;6: 196-204. Bassleer C, Henrotin Y, F ranchimont P. In-vitro evaluation of drugs proposed as chondroprotective agents. IntJ Tissue React 1992;14:231-41. Bassleer C, Rovati L, Franchimont P. Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritis articular cartilage in vitro. Osteoarthritis Cartilage 1998;6:427-34. Bau B, Gebhard P, Haag J, et al. Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro. Arthritis Rheum 2002;46:2648-57. Bayliss MT, Howat S, Davidson C, et al. The organization of aggrecan in human articular cartilage. Evidence for age-related changes in the rate of aggregation of newly synthesized molecules. J Biol Chem 2000;275:6321-7. 78 Bayliss MT, Osborne D, Woodhouse S, et al. Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition. J Biol Chem 1999;274: 15892-900. Beluche LA, Bertone AL, Anderson DE, et al. Effects of oral phenylbutazone to horses on in vitro articular cartilage metabolism. Am J Vet Res 2001 ;62: 1916-21 Benbow U, Brinckerhoff CE. The AP-l site and MMP gene regulation: what is all the fuss about? Matrix Biol 1997;15:519-26. Billinghurst RC, Dahlberg L, Ionescu M et al. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest 1997;99: 1534-45. Bird J L, May S, Bayliss MT. Nitric oxide inhibits aggrecan degradation in explant cultures of equine articular cartilage. Equine VetJ 2000;32:133-9. Bobacz K, Soleiman A, Erlacher L, Smolen J, Graninger WB. Chondrocyte number and proteoglycan synthesis in the ageing and osteoarthritic human articular cartilage. Arthritis Res Ther 2003;5 (Suppl l):94. Boileau C, Martel-Pelletier J, Moldovan F et al. The in situ up-regulation of chondrocyte interleukin-l-converting enzyme and interleukin-18 levels in experimental osteoarthritis is mediated by nitric oxide. Arthritis Rheum 2002;10:2637-47. Bond M, Baker AH, Newby AC. Nuclear Factor KB activity is essential for matrix metalloproteinase-l and —3 upregulation in rabbit dermal fibroblasts. Biochem Biophys Res Commun 1999;264:561-7. Bond M, Fabunmi RP, Baker AH, Newby AC. Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B. FEBS Lett 1998;435:29-34. Borden P, Heller RA. Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases. Crit Rev Eukaryot Gene Exp 1997;7:159-78. 79 Borghaei H, Borghaei RC, Ni X, et al. Evidence that suppression of IL-1 induced collagenase mRNA expression by dihomo-gamma-linolenic acid (DGLA) involves inhibition of NF kappa B binding. Inflamm Res 1997;46 (Suppl 2):S177-8. Bourgeois P, Chales G, Dehais J et al. Efficacy and tolerability of chondroitin sulfate 1200mg/day vs chondroitin sulfate 3 x 400mg/day vs placebo. Osteoarthritis Cartilage l998;6 (Suppl A):25-30 Brama PA, van den Boom R, DeGroot J, et al. Collagenase-l (MMP 1) activity in equine synovial fluid: influence of age, joint pathology, exercise and repeated arthrocentesis. Equine Vet J 2004;36:34-40. Brama PA, TeKoppele J M, Bank RA, et al. Influence of site and age on biochemical characteristics of the collagen network of equine articular cartilage. Am J Vet Res 1999;60:341-5. Brama PA, Tekoppele J M, Bank RA, et al. Influence of different exercise levels and age on the biochemical characteristics of immature equine articular cartilage. Equine VetJSupp 1999;31:55-61. Brama PA, Tekoppele J M, Bank RA, et al. The influence of strenuous exercise on collagen characteristics of articular cartilage in Thoroughbreds age 2 years. Equine Vet J2000;32:551-4. Brama PA, TeKoppele J M, Beekman B, et al. Matrix meta110proteinase activity in equine synovial fluid: influence of age, osteoarthritis, and osteochondrosis. Ann Rheum Dis 1998;57:697-9. Brew K, Dinakarpandian, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 2000;1477z267-83. Bruyere O, Honore A, Ethgen O, et al. Correlation between radiographic severity of knee osteoarthritis and future disease progression. Results from a 3-year prospective, placebo-controlled study evaluating the effect of glucosamine sulfate. Osteoarthritis Cartilage 2003 ;1 1 :1-5. Buckwalter J A. Articular cartilage injuries. Clin Orthop 2002;402:21-37. 80 Bunning RA, Russell RG, Van Damme J. Independent induction of interleukin 6 and prostaglandin E2 by interleukin 1 in human articular chondrocytes. Biochem Biophys Res Commun 1990;166:1163-70. Burns K, J anssens S, Brissoni B, et al. Inhibition of interleukin-l/Toll like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK4. J Exp Med 2003;197:263-8. Burns K, Martinon F, Esslinger C et al. MyD88, an adaptor protein involved in interleukin-1 signaling. J Biol Chem 1998;271:12203-9. Busci L, Poor G. Efficacy and tolerability of oral chondroitin sulfate as a symptomatic slow-acting drug for osteoarthritis (SYSADOA) in the treatment of knee osteoarthritis. Osteoarthritis Cartilage 199816 (Supp A):31-6 Byron CR, Orth MW, Venta PJ, Lloyd J W, Caron JP. Influence of glucosamine on matrix metalloproteinase expression and activity in lipopolysaccharide-stimulated equine chondrocytes. Am J Vet Res 2003;64:666-71. Campo G, Avenoso A, Campo S et al. Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment. Free Radic Res 2003;37: 257-68. Campo G, Avenoso A, Campo S et al. Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats. Arthritis Res Ther 2003;5:122-31. Canapp SO, McLaughlin RM, Hoskinson J J et al. Scintigraphic evaluation of glucosamine hydrochloride and chondroitin sulfate as treatment for acute synovitis in dogs. Am J Vet Res 1999;60:1552-7. Cao M, Westerhausen-Larson A, Niyibizi C et al. Nitric oxide inhibits the synthesis of type-II collagen without altering C012A1 mRNA abundance: prolyl hydroxylase as a possible target. Biochem J 1997;324:305-10. Caron JP. Biology and Pathobiology of Synovial Joints. In Equine Surgery, 2nd edition, Auer J and Stick J, eds. WB Saunders, Philadelphia 1999;pp 665-78. 81 Caron JP, Bowker RM, Abhold RH, et al. Substance P in the synovial membrane and fluid of the equine middle carpal joint. Equine Vet J 1992;24:364-6. Caron JP, Eberhart SW, Nachreiner R. Influence of polysulfated glycosaminoglycan on equine articular cartilage in explant culture. Am J Vet Res 1991;52:1622-5. Caron JP, Fernandes JC, Martel-Pelletier J, et al. Chondroprotective effects of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-l expression. Arthritis Rheum 1996;39:1535-44. Caron JP, Kaneene JB, Miller R. Results of a survey of equine practitioners on the use and perceived efficacy of polysulfated glycosaminoglycan. J Am Vet Med Assoc 1996;209:1564-8. Caron JP, Peters TL, Hauptman JG, et al. Serum concentrations of keratan sulfate, osteocalcin and pyridinoline crosslinks after oral administration of glucosamine to Standardbred horses during race training. Am J Vet Res 2002;63:1106-10. Caron JP, Tardif G, Martel-Pelletier J, et al. Modulation of matrix metalloprotease l3 (collagenase 3) gene expression in equine chondrocytes by IL-1 and corticosteroids. Am J Vet Res 1996;57:1631-4. Caron JP, Toppin DS, Block JA. Effect of polysulfated glycosaminoglycan on osteoarthritic equine articular cartilage in explant culture. Am J Vet Res 1993;54:1116-21. Cawston T, Billington C, Cleaver C, et al. The Regulation of MMPS and TIMPS in cartilage turnover. Ann N Y Acad Sci 1999;878:120-9. Chin JE, Horuk R. IL-1 receptor on rabbit articular chondrocyte: relationship between biological activity and receptor binding kinetics. FASEBJ 1990;4:1481-7. Cho S, Sim J, Jeong C et al. effects of low molecular weight chondroitin sulfate on type II collagen-induced arthritis in DBA/lJ mice. Biol Pharm Bull 2004;27:47-51. 82 Choi DW, Kim MJ, Kin HS et al. A size-exclusion HPLC method for the determination of sodium chondroitin sulfate in pharmaceutical preparations. J Pharm Biomed Anal 2003;31 : 1229-36. Christgau S, Henrotin Y, Tanko L, et al. Osteoarthritic patients with high cartilage turnover Show increased responsiveness to the cartilage protecting effects of glucosamine sulphate. Clin Exp Rheumatol 2004;22:36-42. Clayton HM, Almedia PE, Prades M, et al. Double blind study of the effects of an oral supplement intended to support joint health in horses with tarsal degenerative joint disease. Proceedings American Assoc Equine Pract 2002, p314-7. Clegg PD, Carter SD. Matrix metalloproteinase-2 and —9 are activated in joint disease. Equine Vet J 1999;31:324-30. Clegg PD, Coughlan AR, Carter SD. Equine TIMP l and TIMP-2: identification, activity and cellular sources. Equine VetJ 1998;30:416-23 Clegg PD, Coughlan AR, Riggs CM, et al. Matrix metalloproteinases 2 and 9 in equine synovial fluids. Equine VetJ 1997;29:343-8. Clegg PD, Jones MD, Carter SD. The effect of drugs commonly used in the treatment of equine articular disorders on the activity of equine matrix metalloproteinase-2 and 9. J Vet Pharmacol Therap 1998;21:406-13. Chung JY, Chul Park Y, Ye H, et al. All TRAFS are not created equal: common and distinct molecular mechanisms of TRAP-mediated signal trandsduction. J Cell Sci 2002:115;679-88. Colasanti M, Persichini T, Menegazzi M, et al. Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide. J Biol Chem 1995 ;270:2673 1-3. Cole AA, Kuettner KE. MMP-8 (neutrophil collagenase) mRNA and aggrecanase cleavage products are present in normal and osteoarthritic human articular cartilage. Acta Ortop Scand Suppl 1995;266:98-102. 83 Cole AA, Chubinskaya S, Schumacher B, et al. Chondrocyte matrix metalloproteinase-8. Human articular chondrocytes express neutrophil collagenase. JBiol Chem 1996; 271:11023-6. Conte A, de Bernardi M, Palmieri L, et al. Metabolic fate of exogenous chondroitin sulfate in man. Arzneim-Forsch. Drug Res 1991;41:768-72. Conte A, Volpi N, Palmieri L, et al. Biochemical and pharmacokinetic aspects of oral treatment with chondroitin sulfate. Arzneim-Forsch. Drug Res 1995;45:918-25. Conrozier T. Death of articular chondrocytes. Mechanisms and protection. Presse Med 1998;27:1859-61. Cook JL, Anderson C, Kreeger JM, et al. Effects of human recombinant interleukin-10 on canine articular chondrocytes in three-dimensional culture. Am J Vet Res 2000;7z766-70. Dart AJ, Perkins N, Dowling BA et al. The effect of three different doses of sodium pentosan polysulfate on haematological and haemostatic variables in adult horses. Aust VetJ2001;79:624-7 Das A, Hammad TA. Efficacy of a combination of FCHG49 glucosamine hydrochloride, TRH122 low molecular weight sodium chondroitin sulfate and manganses ascorbate in the management of knee osteoarthritis. Osteoarthritis Cartilage 2000;8:343-50. Davidson G. Pharm Profile: Glucosamine and chondroitin sulfate. Compend Cont Educ Pract Vet 2000;454-8. de Mattei M, Pellati A, Pasello M, et al. High doses of glucosamine-HCI have detrimental effects on bovine articular cartilage explants cultured in vitro. Osteoarthritis Cartilage 2002; 10:816-25. Dean DD. Proteinase-mediated cartilage degradation in osteoarthritis. Semin Arthritis Rheum 1991;20:2-11. 84 Dean DD, Azzo W, Martel-Pelletier J et al. Levels of metalloproteases and tissue inhibitor of metalloproteases in human osteoarthritic cartilage. J Rheumatol 1987;14:43-4. Dechanet J, Taupin JL, Chomarat P, et al. Interleukin-4 but not interleukin-10 inhibits the production of leukemia inhibitory factor by rheumatoid synovium and synoviocytes. Eur J Immunol 1994;24:3222-8. Del Carlo M, Loeser RF. Nitric oxide-mediated chondrocyte cell death requires the generation of additional reactive oxygen species. Arthritis Rheum 2002;46:394-403. DiBattista JA, Pelletier JP, Zafarullah M, et al. Interleukin-1 beta induction of tissue inhibitor of metalloproteinase (TIMP 1) is functionally antagonized by prostaglandin E2 in human synovial fibroblasts. J Cell Biochem 1995;57:619-29. Dingle J T. The effect of nonsteroidal antiinflammatory drugs on human articular cartilage glycosaminoglycan synthesis. Osteoarthritis Cartilage 1999;7z3 13-4. Dodge GR, Jimenez SA. Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes. Osteoarthritis Cartilage 2003;11:424-32. Dodge GR, Regatte RR, Hall J O et al. The fate of oral glusocamine traced by 13C- labelling in the dog. Abstract, American College Radiology meeting, San Francisco, California, November 2001. Dorna VJ, Guerrero RC. Effects of oral and intramuscular use of chondroitin sulphate in induced equine aseptic arthritis. J Equine Vet Sci 1998;18:548-55. Du J, Eddington N. Determination of the chondroitin sulfate disaccharides in dog and horse plasma by HPLC using chondroitinase digestion, precolumn derivatization, and fluorescence detection. Anal Biochem 2002;306:252-58. Du J, White N, Eddington ND. The bioavailability and pharmacokinetics of glucosamine hydrochloride and chondroitin sulfate after oral and intravenous single dose administration in the horse. Biopharm Drug Dispos 2004; 25:109-16. 85 Eddington ND, Du J, White N. Evidence of the oral absorption of chondroitin sulfate as determined by total disaccharide content after oral and intravenous administration to horses. Proceedings Am Assoc Equine Pract 2001;47:326-8 . El-Saharty YS, Abdel Bary A. High performance liquid chromatographic determination of nutraceuticals, glucosamine sulphate and chitosan, in raw materials and dosage forms. Analytica Chimica Acta 2002;125-31. Fenton J I, Chlebek KA, Caron JP, et al. Effect of glucosamine on interleukin-1 conditioned articular cartilage. Equine VetJ 2002;Supp 34:219-23. Fenton J I, Chlebek-Brown KA, Peters TL, et al. Glucosamine HCl reduces equine articular cartilage degradation in explant culture. Osteoarthritis Cartilage 2000;8:258-65. Fenton JI, Chlebek-Brown KA, Peters TL, et al. The effects of glucosamine derivatives on equine articular cartilage degradation in explant culture. Osteoarthritis Cartilage 2000;8 :444-5 1 . Fenton J I, Orth MW, Chlebek-Brown KA, et al. Effect of longeing and glucosamine supplementation on serum markers of bone and joint metabolism in yearling quarter horses. Can J Vet Res 1999;63:288-91 Fernandes J C, Martel-Pelletier J, Pelletier JP. The role of cytokines in osteoarthritis pathophysiology. Biorheology 2002;39:237-46. Firestein GS, Manning AM. Signal transduction and transcription factors in rheumatic disease. Arthritis Rheum 1999;4:609-21. Flannery CR, Lark MW, Sandy JD. Identification of a stromelysin cleavage Site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage. J Biol Chem 1992;267:1008-14. Francis DJ, Hutadilok N, Kongtawelert P, et al. Pentosan polysulphate and glycosaminoglycan polysulphate stimulate the synthesis of hyaluronan in vivo. Rheumatol Int 1993;13:61-4. 86 Frean SP, Bryant CE, F roling IL, et al. Nitric oxide production by equine articular cartilage cells in vitro. Equine VetJ 1997;29:98-102. Frean SP, Lees P. Effects of polysulfated glycosaminoglycan and hyaluronan on prostaglandin E2 production by cultured equine synoviocytes. Am J Vet Res 2000;61:499-505. Frisbie DD, Nixon AJ. Insulin-like growth factor 1 and corticosteroid modulation of chondrocyte metabolic and mitogenic activities in interleukin 1-conditioned equine cartilage. Am J Vet Res 1997;58:524-30. Frisbie D, Ricky E, Southwood L, McIlwraith W, Colhoun H. Evaluation of pentosan polysulfate. Orthopedic Research Laboratory, Colorado State University 2003;1-4. Frisbie DD, Sandler EA, Trotter GW, et al. Metabolic and mitogenic activities of insulin-like growth factor-1 in interleukin-l conditioned equine cartilage. Am J Vet Res 2000;61:436-41. Fuller CJ, Barr AR, Sharif M, et al. Cross-sectional comparison of synovial fluid biochemical markers in equine osteoarthritis and the correlation of these markers with articular cartilage damage. Osteoarthritis Cartilage 2001;9:49-55. Fuller CJ, Ghosh P, Barr AR. Plasma and synovial fluid concentrations of calcium pentosan polysulphate achieved in the horse following intramuscular injection. Equine Vet J 2002;34:61-4. Gaustad G, Larsen S. Comparison of polysulphated glycosaminoglycan and sodium hyaluronate with placebo in treatment of traumatic arthritis in horses. Equine Vet J 1995;27:356-62. Geng Y, Valbracht J, Lotz M. Selective activation of the Mitogen-activated Protein Kinase Subgroups c-Jun NH2 Terminal Kinase and p38 by IL-1 and TNF in Human Articular Chondrocytes. J Clin Invest 1996; 98:2425-30. Ghosh P. The role of hyaluronic acid (hyaluronan) in health and disease: interactions with cells, cartilage and components of synovial fluid. Clin Exp Rheumatol 1994;12:75-82. 87 Ghosh P. The pathobiology of osteoarthritis and the rationale for the use of pentosan polysulfate for its treatment. Semin Arthritis Rheum 1999;28:211-67. Ghosh P, Hutadilok N. Interactions of pentosan polysulfate with cartilage matrix proteins and synovial fibroblasts derived from patients with osteoarthritis. Osteoarthritis Cartilage 1996;4z43-53 Gibson KT, Hodge H, Whittem T. Inflammatory mediators in equine synovial fluid. Aust VetJ 1996; 73:148-51. Glade MJ. Polysulfated glycosaminoglycan accelerates net synthesis of collagen and glycosaminoglycans by arthritic equine cartilage tissues and chondrocytes. Am J Vet Res 1990;51:779-85. Goldberg HJ, Scholey J, Fantus IG. Glucosamine activates the plasminogen activator inhibitor 1 gene promoter through Spl binding sites in glomerular mesangial cells. Diabetes 2000;49:863-71. Goldring MB. The role of the chondrocyte in osteoarthritis. Arthritis Rheum 2000;43:1916-26. Goldring MB, Birkhead J, Sandell LJ, et al. Interleukin 1 suppresses expression of cartilage-specific types 11 and IX collagens and increases types I and III collagens in human chondrocytes. J Clin Invest 1988;82:2026-37. Goldring MB, Birkhead JR, Suen LF, et al. Interleukin-1 beta modulated gene expression in immortalized human chondrocytes. J Clin Invest 1994;94:2307-16. Gouze JN, Bordji K, Gulberti S, et al. Interleukin-113 down-regulates the expression of Glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: Influence of Glucosamine on Interleukin-IB-medicated effects in rat chondrocytes. Arthritis Rheum 2001 :44:351-60. Gouze JN, Bianchi A, Bécuwe P, et al. Glucosamine modulates IL-l-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-KB pathway. F EBS Lett 2002:510;166-70. 88 Grabowski PS, Wright PK, Van’t Hof RJ et al. Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis. Br J Rheumatol l7;36:651-5. Gustafson SB, McIlwraith CW, Jones RL. Comparison of the effect of polysulfated glycosaminoglycan, corticosteroids, and sodium hyaluronate in the potentiation of a subinfective dose of Staphylococcus aureus in the midcarpal joint of horses. Am J Vet Res 1989;50:2014-7. Gustafson SB, McIlwraith CW, Jones RL, et al. Further investigations into the potentiation of infection by intra-articular injection of polysulfated glycosaminoglycan and the effect of filtration and intra-articular injection of amikacin. Am J Vet Res 1989;50:2018-22. Hambach L, Neureiter D, Zeiler G, et al. Severe disturbance of the distribution and expression of type VI collagen chains in osteoarthritic articular cartilage. Arthritis Rheum 1998;41:986-96. Hamm D, Goldman L, Jones EW. Polysulfated glycosaminoglycan: a new intra- articular treatment for equine lameness. Vet Med 1984;79:811-6. Han Z, Boyle DL, Manning AM, et al. AP-l and NF-kappaB regulation in rheumatoid arthritis and murine collagen-induced arthritis. Autoimmunity 1998:28zl97-208. Hanson RR. Oral glycosaminoglycans in treatment of degenerative joint disease in horses. Equine Pract 1996;18:18-22. Hanson RR, Brawner WR, Blaik MA et al. Oral treatment with a nutraceutical (Cosequin) for ameliorating signs of navicular syndrome in horses. Vet Therapeutics 2001 ;2: 148-59. Hanson R, Smalley LR, Huff GK, et al. Oral treatment with a glucosamine- chondoitin sulfate compound for degenerative joint disease in horses:25 cases. Equine Pract 1997;19: 16-22. Hardingham T. Chondroitin sulfate and joint disease. Osteoarthritis Cartilage 1998;6 (Suppl A):3-5. 89 Hayashi T, Abe E, Yamate T, et al. Nitric oxide production by superficial and deep articular chondrocytes. Arthritis Rheum 1997;40:261-9. Hilbert BJ, Rowley G, Antonas KN, et al. Changes in the synovia after the intra- articular injection of sodium hyaluronate into normal horse joints and after arthrotomy and experimental cartilage damage. Aust VetJ 1985;62:182-4. Hoffer LJ, Kaplan LN, Hamadeh MJ et al. Sulfate could mediate the therapeutic effect of glucosamine sulfate. Metabolism 2001;50:767-70. Howard RD, McIlwraith CW. Hyaluronan and its use in the treatment of equine joint disease. In: McIlwraith CW, Trotter GW, eds. Joint Disease in the Horse. WB Saunders, Philadelphia 1996;257-69. Howard RD, McIlwraith CW, Trotter GW, et al. Cloning of IL-1 alpha and equine interleukin 1 beta and determination of their full length cDNA sequences. Am J Vet Res 1998;59:704-11. Hua J, Sakamoto K, Nagaoka I. Inhibitory actions of glucosamine, a therapeutic agent for osteoarthritis, on the function of neutrophils. J Leukoc Biol 2002;71:632-40. Huang Y, Toyoda H, Toida T, et al. Determination of chondroitin sulfates in human whole blood, plasma and blood cells by high-performance liquid chromatography. Biomed Chromatogr 1995;9:102-5. Hughes R and Carr A. A randomized double blind, placebo controlled trial of glucosamine sulphate as an analgesic in osteoarthritis of the knee. Rheumatol 2002; 41:279-84. Hulse DS, Hart D, Slatter M, et al. The effect of Cosequin in cranial cruciate deficient and reconstructed stifle joints in dogs. In: Proceedings 25th Annual Conference Veterinary Orthopedic Society 1998;64. 90 Ilic MZ, Martinac B, Handley CJ. Effects of long—term exposure to glucosamine and mannosamine on aggrecan degradation in articular cartilage. Osteoarthritis Cartilage 2003;1 1:613-22. Innes J F, Barr AR, Sharif M. Efficacy of oral calcium pentosan polysulfate for the treatment of osteoarthritis of the canine stifle joint secondary to cranial cruciate ligament deficiency. Vet Rec 2000;146:433-7 .Ismaiel S, Atkins RM, Pearse MF et al. Susceptibility of normal and arthritic human articular cartilage to degradative stimuli. Br J Rheumatol 1992;31:369-73. Janssens S, Burns K, Vercammen E, et al. MyD88S, a splice variant of MyD88, differentially modulates NflrappaB and AP-l dependent gene expression. FEBS Lett 2003 ;548: 103-7. Jiang Z, Ninomiya-Tsuji J, Qian Y et al. Interleukin-1 (IL-l) receptor-associated kinase-dependent IL-l induced signaling complexes phosphorylate TAKl and TABZ at the plasma membrane and activate TAKl in the cytosol. Mol Cell Biol 2002;22:7158-67. Jeffcott LB, Rossdale PD, Freestone J, et al. An assessment of wastage in Thoroughbred racing from conception to 4 years of age. Equine Vet J 1992;14:185- 98. Jimenez SA, Dodge GR. The effects of glucosamine sulfate on human chondrocyte gene expression. Osteoarthritis Cartilage 1997;5 (Supp A):72. Johnson KA, Hulse DA, Hart RC, et al. Effects of an orally administered mixture of chondroitin sulfate, glucosamine hydrochloride and manganese ascorbate on synovial fluid chondroitin sulfate 3B3 and 7D4 epitope in a canine cruciate ligament transection model of osteoarthritis. Osteoarthritis Cartilage 2001;9:14-21. Jolly WT, Whittem T, Jolly AC, et al. The dose-related effects of phenylbutazone and a methylprednisolone acetate formulation (Depo-Medrol) on cultured explants of equine carpal articular cartilage. J Vet Pharmacol Ther 1995;18:429-37. Kahle P, Saal JG, Schaudt K, et al. Determination of cytokines in synovial fluid: correlation with diagnosis and histomorphological characteristics of synovial tissue. Ann Rheum Dis 1992;51:731-4. 91 Kaneene JB, Ross WA, Miller R. The Michigan equine monitoring system. 11. Frequencies and impact of selected health problems. Prev Vet Med 1997; 29, 277-92. Kashiwagi M, Tortorella M, Nagase H, et al. TIMP 3 is a potent inhibitor of aggrecanase 1 (ADAM-T84) and aggrecenase 2 (ADAM-T85). J Biol Chem 2001;276:12501-4. Kato H, Ohashi T, Nakamura N et al. Molecular cloning of equine interleukin-l alpha and beta CDNAS. Vet Immunol Immunopathol 1995;48:221-31 Kawcak CE, Frisbie DD, Trotter GW, et al. Effects of intravenous administration of sodium hyaluronate on carpal joints in exercising horses after arthroscopic surgery and osteochondral fragmentation. Am J Vet Res 1997;58:1132-40. Kelly GS. The role of glucosamine sulfate and chondroitin sulfates in the treatment of degenerative joint disease. Altern Med Rev 1998;3 :27-39. Kim J J, Conrad HE. Effect of D-glucosamine concentration on the kinetics of mucopolysaccharide biosynthesis in cultured chick embryo vertebral cartilage. J Biol Chem 1974;249:3091-7. Kinoshita A, Sugahara K. Microanalysis of glycosaminoglycan-dcrived oligosaccharides labeled with a fluorophore 2-aminobemzamide by High- Performance Liquid Chromatography: Application to disaccharide composition analysis and exosequencing of oligosaccharides. Anal Biochem 1999;269:367-78. Kirker-Head CA, Chandna VK, Agarwal RK, et al. Concentrations of substance P and prostaglandin E2 in synovial fluid of normal and abnormal joints of horses. Am J Vet Res 2000;6z714-8. Kirker-Head CA, Kirker-Head RP. Safety of an oral chondroprotective agent in horses. Vet Therapeutics 2001;2z345-53. Kopp E, Medzhitov R, Carothers J et al. ECSIT is an evolutionary conserved intermediate in the Toll/IL-l signal transduction pathway. Genes Dev 1999;13:2059- 71. Koshy PJT, Lundy CJ, Rowan AD, et al. The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by 92 interleukin-1 and oncostatin M: A time course study using real time Quantitative Reverse Transcription- Polymerase Chain Reaction. Arthritis Rheum 2002;46:961-7. Kovalovsky D, Refojo D, Holsboer F, Arzt E. Molecular mechanisms and Th1/Th2 pathways in corticosteroid regulation of cytokine production. Neuroimmunol 2000;109:23-9. Krane SM. Some molecular mechanisms of glucocorticoid action. Br J Rheumatol 1993;32 (Suppl 2):3-5. Kummer JA, Abbink J J , de Boer JP, et al. Analysis of intraarticular fibrinolytic pathways in patients and noninflammatory diseases. Arthritis Rheum 1992;35:884-93. Largo R, Alvarez-Soria MA, Diez-Ortego I, et al. Glucosamine inhibits IL-IB- induced NFKB activation in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 2003 :1 1 ;290-8. Lark MW, Bayne EK, Flanagan J, et al. Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. J Clin Invest 1997;100:93-106. Lauder RM, Huckerby TN, Brown GM, et al. Age-related changes in the sulphation of the chondroitin sulphate linkage region from human articular cartilage aggrecan. Biochem J2001;358:523-8. Laverty S, Sandy JD, Celeste C, et al. Synovial fluid levels and serum pharmacokinetics in a large animal model following treatment with oral glucosamine at clinically relevant doses. Arthritis Rheum 2005;52:181-91. Leffler CT, Philippi AF, Leffler SG, et al. Glucosamine, chondroitin, and manganese ascorbate for degenerative joint disease of the knee or low backza randomized, double blind, placebo controlled pilot study. Mil Med 1999;164:85-91. Li X, Commane M, Jiang Z et a1. IL-l-induced NflrappaB and c-Jun N-terminal kinase (JN K) activation diverge at IL-l receptor-associated kinase (IRAK). Proc Nat Acad Sci USA 2001;98:4461-5. 93 Liang Z, Leslie J, Adebowale A, et al. Determination of the nutraceutical, glucosamine hydrochloride, in raw materials, dosage forms and plasma using pre- column derivatization with ultraviolet HPLC. J Pharm Biomed Anal 1999;20:807-14. Lippiello L, Woodward J, Karpman R, Hammad A. In vivo chondroprotection and metabolic synergy of glucosamine and chondroitin sulfate. Clin Orthop Ret Res 2000;381 :229-40 Lippiello L, Han MS, Henderson T. Protective effect of the chondroprotective agent Cosequin® on bovine articular cartilage exposed in vitro to nonsteroidal anti- inflammatory agents. Vet Therapeutics 2002;2:128-35. Lippiello L. Glucosamine and chondroitin sulfate: biological response modifiers of chondrocytes under simulated conditions of joint stress. Osteoarthritis Cartilage 2003;] 1:335-42. Little CB, Flannery CR, Hughes CE et al. Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro. Biochem J 1999 15;344:61-8. Little CB, Hughes CE, Curtis CL et al. Matrix metalloproteinases areinvolved in C- terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation. Matrix Biol 2002;21:271-88. Lippiello L, Idouraine A, McNamarea PS et al. Cartilage stimulatory and antiproteolytic activity is present in sera of dogs treated with a chondroprotective agent. Canine Pract 1999;24:18-9. Lohmander LS, Neame PJ, Sandy JD. The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum 1993 ;36:1214-22. Lotz M, Guerne PA. Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-l/erythroid potentiating activity. J Biol Chem 1991;266:2017-20. Lualdi P. Bioavailability of oral chondroitin sulfate. Rheumatol 1993;13:39-40. 94 Ma L, Rudert WA, Harnaha J, et al. Immunosuppressive effects of glucosamine. J Biol Chem 2002;277:39343-9. MacDonald MH, Stover SM, Willits NH, et al. Regulation of matrix metabolism in equine cartilage explant cultures by interleukin 1. Am J Vet Res 1992;53:2278-85. MacDonald MH, Stover SM, Willits NH, et al. Effect of bacterial lipopolysaccharides on sulfated glycosaminoglycan metabolism and prostaglandin E2 synthesis in equine cartilage explant cultures. Am J Vet Res 1994;55:1127-38. Maier R, Ganu V, Lotz M. Interleukin-11, an inducible cytokine in human articular chondrocytes and synoviocytes, stimulates the production of the tissue inhibitor of metalloproteinases. J Biol Chem 1993;268: 21527-32. Malone ED. Managing chronic arthritis. Vet Clin North Am Equine Pract 2002;18:411-37. Mankin HJ, Radin EL. Structure and Function of joints. In: Koopman WJ, ed. Arthritis and Allied Conditions: A textbook of Rheumatology Williams and Wilkins, Baltimore 1997. Martel-Pelletier J. PathophySiology of osteoarthritis. Osteoarthritis Cartilage 1998;6:374-376. Martel-Pelletier J, Alaaeddine N, Pelletier JP. Cytokines and their role in the pathophysiology of osteoarthritis. Front Biosci l999;4:D694-703. Martel-Pelletier J, Cloutier JM, Pelletier JP. Neutral proteases in human osteoarthritic synovium. Arthritis Rheum 1986;29:1112-21. Martel-Pelletier J, McCollum R, DiBattista J A, et al. The interleukin-1 receptor in normal and 0A human articular chondrocytes is the type-1 receptor: Binding kinetics and biological function. Arthritis Rheum 1992;35:530-40 95 Martel-Pelletier J, McCollum R, F ujimoto N et al. Excess of metalloproteases over tissue inhibitors of metalloproteases may contribute to cartilage degradation in OA and rheumatoid arthritis. Lab Invest 1994;70:807-15. Martel-Pelletier J, Mineau F, Jovanovic D, et al. MAPK and NF-KB together regulate the IL-l7-induced nitric oxide production in human OA chondrocytes: possible role of transactivating factor MAPKAP-K. Arthritis Rheum 1999;42:2399- 409. Martin JA, Buckwalter JA. The role of chondrocyte senescence in the pathogenesis of osteoarthritis and in limiting cartilage repair. J Bone Joint Surg Am 2003;85-A (Suppl 2):106-10. Matheu V, Garcia Bara MT, Pelta R, et al. Immediate hypersensitivity reaction to glucosamine sulfate. Allergy 1999;54:643. May SA, Hooke RE, Lees P. The effect of drugs used in the treatment of osteoarthrosis on stromelysin (proteoglycanase) of equine synovial cell origin. Equine VetJ Suppl 1988;6128-32. May SA, Hooke RE, Lees P. The characterization of equine interleukin-1. Vet Immunol Immunopathol 1990;24: 169-75. May SA, Hooke RE, Lees P. Interleukin-1 stimulation of equine articular cells. Res Vet Sci 1992;52:342-348. May SA, Hooke RE, Lees P. Species restrictions demonstrated by the stimulation of equine cells with recombinant human interleukin-1. Vet Immunol Immunopathol 1992;30:373-384. McAlindon TE, LaValley MP, Gulin JP, et al. Glucosamine and chondroitin for treatment of osteoarthritisza systematic quality assessment and meta-analysis. JAMA 2000; 283 : 1469-1475. McCarthy G, Seed M, Barabas S, et al. Clinical Trial comparing glucosamine with chondroitin sulfate to carprofen for the treatment of osteoarthritis in dogs- preliminary findings. J Vet Int Med 2003;17:426. 96 McIlwraith CW. Current concepts in equine degenerative joint disease. J Am Vet Med Assoc 1982;180:239-50. McIlwraith CW. General pathobiology of the joint and response to injury. In: McIlwraith CW, Trotter GW, eds. Joint Disease in the Horse WB Saunders, Philadelphia 1996:40-70. McIlwraith CW. Use of sodium hyaluronate (Hyaluronan) in equine joint disease. Equine Vet Educ 1997;9z296-304. McNamara PS, Barr SC, Erb HN. Hematologic, hemostatic and biochemical effects in dogs receiving an oral chondroprotective agent for thirty days. Am J Vet Res 1996;57:1390-4. McNamara PS, Barr SC, Erb HN, et al. Hematological, hemostatic, and biochemical effects in cats receiving an oral chondroprotective agent for thirty days. Vet Therapeutics 2000;] : 108-17. Mendes AF, Caramona MM, Carvalho AP, et al. Differential roles of hydrogen peroxide and superoxide in mediating IL-l induced NF-kappaB activation and iNOS expression in bovine articular chondrocytes. J Cell Biochem 2003;88:783-93. Mendes AF, Carvalho AP, Caramona MM, et al. Role of nitric oxide in the activation of NF-kappa B, AP-l and NOS II expression in articular chondrocytes. Inflamm Res 2002;51:369-75. Mello DM, Nielsen BD, Peters et al. Comparison of inhibitory effects of glucosamine and mannosamine on bovine articular cartilage degradation in vitro. Am J Vet Res 2004;65: 1440-5. Mengshol JA, Mix KS, Brinckerhoff CE. Matrix metalloproteinases as therapueutic targets in arthritic diseases: bull’s-eye or missing the mark? Arthritis Rheum 2002;46: 13-20. 97 Mengshol JA, Vincenti MP, Brinckerhoff CE. IL-l induces collagenase-3 (MMP 13) promoter activity in stably transfected chondrocytic cells: requirement for Runx-2 and activation by p38 MAPK and JNK pathways. Nuc Acids Res 2001;29:4361-72. Mengshol JA, Vincenti MP, Coon CI, et al. Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-JUN N-terminal kinase and nuclear factor KB: Differential regulation of collagenase 1 and 3. Arthritis Rheum 2000;43:801-11. Miagkov AV, Kovalenko DV, Brown CE, et al. NF-KB activation provides the potential link between inflammation and hyperplasia in the arthritic joint. Proc Nat Acad Sci 1998;95:13859-64. Milner J M, Elliott SF, Cawston TE. Activation of procollagenases is a key control point in cartilage collagen degradation: Interaction of serine and metalloproteinase pathways. Arthritis Rheum 2001;9z2084-96. Mitchell PG, Magna HA, Reeves LM, et al. Cloning, expression and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest 1996;97 :761-8. Monauni T, Zenti MG, Cretti A, et al. Effects of glucosamine infusion on insulin secretion and insulin action in humans. Diabetes 2000;49:926-35. Morreale P, Manopulo R, Galati M et al. Comparison of the anti-inflammatory efficacy of chondroitin sulfate and diclofenac sodium in patients with knee osteoarthritis. J Rheumatol 1996;23:1385-91 Morris EA, MacDonald BS, Webb AC, et al. Identification of interleukin-1 in equine osteoarthritic joint effusions. Am J Vet Res 1990;51:59-64. Morris EA, Treadwell BV. Effect of interleukin 1 on articular cartilage from young and aged horses and comparison with metabolism of osteoarthritic cartilage. Am J Vet Res 1994;55:138-46. 98 Morris EA, Wilcon S, Treadwell BV. Inhibition of interleukin l-mediated proteoglycan degradation in bovine articular cartilage explants by addition of sodium hyaluronate. Am J Vet Res 1992;53:1977-82. Moses VS, Hardy J, Bertone AL, et al. Effects of anti-inflammatory drugs on lipopolysaccharide-challenged and —unchallenged equine synovial explants. Am J Vet Res 2001;62:54-60. Moses VS. Bertone AL. Nonsteroidal anti-inflammatory drugs. In Pain Management and Anesthesia, Vet clinics North Am Equine Pract 2002;18:21-38. Moses VS, Hardy J, Bertone AL, et al. Effects of anti-inflammatory drugs on lipopolysaccharide-challenged and -unchallenged equine synovial explants. Am J Vet Res 2001;62:54-60. Mroz PJ, Silbert J E. Use of 3H-glucosamine and 35S-sulfate with cultured human chondrocytes to determine the effect of glucosamine concentration on formation of chondroitin sulfate. Arthritis Rheum 2004;50:3574-9. Muller-Fassbender H, Bach GL, Haase W, Rovati LC, Setnikar I. Glucosamine sulphate compared to ibuprofen in osteoarthritis of the knee. Ostearthritis Cartilage l994;2:61-9 Muller-Gerbl M, Schulte E, Putz R. The thickness of the calcified layer of articular cartilage: a function of the load supported? J Anat 1987;154:103-11. Munteanu SE, Ilic MZ, Handley CJ. Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures. Matrix Biol 2002;21 :429-40. Murray RC, Birch HL, Lakhani K, et al. Biochemical composition of equine carpal articular cartilage is influenced by Short-term exercise in a site specific manner. Ostearthritis Cartilage 2001:9:625-32. Murray RC, Whitton RC, Vedi S, et al. The effect of training on the calcified zone of equine middle carpal articular cartilage. Equine Vet J 1999 (Supp);30:274-8. 99 Murrell GA, Jang D, Williams RJ. Nitric oxide activates protease enzymes in articular cartilage. Biochem Biophys Res Commun 1995;206:15-21. Nagase H, Visse R. Matrix metalloproteinases and Tissue Inhibitors of Metalloproteinases: Structure, function and biochemistry. Circ Res 2003;92:827-39. Nagase H, Woessner F. Matrix metalloproteinases. Minireview. J Biol Chem 1999;274: 21491-4. Naito K, Takahashi M, Kushida K, et al. Measurement of matrix metalloproteinases (MMPS) and tissue inhibitor of metalloproteinases-1 (TIMP 1) in patients with knee osteoarthritis: comparison with generalized osteoarthritis. Rheumatol 1999;38:510-5. Narita H, Takeda Y, Takagaki K, et al. Identification of glycosaminoglycans using high-performance liquid chromatography on a hydroxyapatite column. Anal Biochem 1995;232:133-6. Nakamura H, Shibakawa A, Tanaka M et al. Effects of glucosamine hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteases by chondrocytes and synoviocytes in osteoarthritis. Clin Exp Rheumatol 2004;22:293-9. Nerucci F, Fioravanti A, Cicero MR, et al. Effects of chondroitin sulfate and interleukin-lbeta on human chondrocyte cultures exposed to pressurization: a biochemical and morphological study. Ostearthritis Cartilage 2000;8:279-87. Newton R, Kuitert LM, Bergmann M, et al. Evidence for the involvement of NF- kappaB in the transcriptional control of COX 2 gene expression by IL-lbeta. Biochem Biophys Res Commun 1997:237128-32. Nietfeld JJ, Wilbrink B, Helle M et al., J. Interleukin-l-induced interleukin-6 is required for the inhibition of proteoglycan synthesis by interleukin-1 in human articular cartilage. Arthritis Rheum 1990;33:1695-701. Ninomiya-Tsuji J, Kishimoto K, Hiyama A, et al. The kinase TAK] can activate the NIK-IKB as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 1999;398: 252-6 100 Nishikawa H, Mori I, Umemoto J. Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane. Arch Biochem Biophys 1985;240:146-53. Noack W, Fischer M, Forster KK et al. Glucosamine sulfate in osteoarthritis of the knee. Ostearthritis Cartilage l994;2:51-9. Noyszewski EA, Wrobleewski K, Dodge GR, et al. Preferential Incorporation of glucosamine into the galactosamine moieties of chondroitin sulfates in articular cartilage explants. Arthritis Rheum 2001;44: 1089-95. Oegema TR Jr, Deloria LB, Sandy JD, et al. Effect of oral glucosamine on cartilage and meniscus in normal and chymopapain-injected knees of young rabbits. Arthritis Rheum 2002; 46:2495-503. ' Oh M, Fukuda K, Asada S, et al. Concurrent generation of nitric oxide and superoxide inhibits PG synthesis in bovine articular chondrocytes; involvement of peroxynitrite. J Rheumatol 1998 :25 :2 1 69-74. Ohuchi E, Imai K, Fujii Y, et al. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem 1997;272:2446-51. Okada Y, Shinmei M, Tanaka O, et al. Localization of matrix metalloproteinase 3 (stromelysin) in osteoarthritic cartilage and synovium. Lab Invest 1992;66:680-90. Omata T, Segawa Y, Itokazu Y, et al. Effects of chondrotin sulfate-C on bradykinin- induced proteoglycan depletion in rats. Arzneimitt 1999;49:577-81. O’Neill LA, Dunne A, Edjeback M, et al. Mal and Myd88: adaptor proteins involved in Signal transduction by Toll-like receptors. J Endotoxin Res 2003;9:55-9. Orth MW, Peters TL, Hawkins JN. Inhibition of articular cartilage degradation by glucosamine-HCI and chondroitin sulfate. Equine Vet J 2002;3 :224-9. 101 Owens JG, Kamerling SG, Stanton SR, et al. Effects of pretreatment with ketoprofen and phenylbutazone on experimentally induced synovitis in horses. Am J Vet Res 1996; 57:866-74. Palmer JL, Bertone AL. Joint Biomechanics in the pathogenesis of traumatic arthritis. In: McIlwraith CW, Trotter GW, eds. Joint Disease in the Horse. WB Saunders, Philadelphia 1996;104-19. Palmer J L, Bertone AL, Litsky AS. Contact area and pressure distribution changes of the equine third carpal bone during loading. Equine Vet J 1994;26:197-202. Palmer JL, Bertone AL, Malemud CJ, et al. Site-specific proteoglycan characteristics of third carpal articular cartilage in exercised and nonexercised horses. Am J Vet Res 1995;56:1570-6. Palmer J L, Bertone AL, Mansour J, et al. Biomechanical properties of third carpal articular cartilage in exercised and nonexercised horses. J Orthop Res 1995;13:854- 60. Palmierei L, Conte A, Giovannini L, Pualdi P and Ronca G. Metabolic fate of exogenous chondroitin sulfate in the experimental animal. Arzneim-Forsch Drug Res 1990;40:319-23 Park SK, Lin HL, Murphy S. Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF -kappaB binding to DNA. Biochem J 1997;322:609-13. Paroli E, Antonilli L, Biffoni M. A pharmacological approach to glycosaminoglycans. Drugs Exp Clin Res 1991;17:9-19. Patti ME, Virkamaki A, Landaker EJ, et al. Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early postreceptor insulin signaling events in skeletal muscle. Diabetes 1999;48:1562-71. Patwari P, Kurz B, Sandy JD, et al. Mannosamine inhibits aggrecanase-mediated changes in the physical properties and biochemical composition of articular cartilage. Arch Biochem Biophys 2000;374:79-85. 102 Pavelka K, Bruyere O, Rovati LC, et al. Relief in mild-to-moderate pain is not a confounder in joint space narrowing assessment of full extension knee radiographs in recent osteoarthritis structure-modifying drug trials. Ostearthritis Cartilage 2003;11:730-7. Pavelka K, Gatterova J, Olejarova M, Machacek S, Giacovetti G, Rovati L. Glucosamine sulfate use and delay- of progression of knee 0A: A 3-year, randomized, placebo-controlled, double blind study. Arch Intern Med 2002;162:21 13- 23 Pelletier JP, DiBattista J A, Raynauld JP, et al. The in vivo effects of intraarticular corticosteroid injections on cartilage lesions, stromelysin, interleukin-1, and oncogene protein synthesis in experimental osteoarthritis. Lab Invest 1995;72:578-86. Pelletier JP, DiBattista JA, Roughley P, et a1. Cytokines and inflammation in cartilage degradation. Rheum Dis Clin North Am 1993;19:545-68. Pelletier JP, Lascau-Coman V, Jovanovic D, et al. Selective inhibition of inducible nitric oxide synthase in experimental osteoarthritis is associated with reduction in tissue levels of catabolic factors. J Rheumatol 1999;26:2002-14. Pelletier JP, Martel-Pellletier J. Evidence for the involvement of IL-1 in human OA cartilage degradation: protective effect of NSAID. J Rheumatol 1989;16 (Supp 18);]9-27. Pelletier JP, Martel-Pelletier J, Howell DS. Etiopathogenesis of osteoarthritis. In Koopman WJ, Ed. Arthritis and Allied conditions. A Textbook of Rheumatology. 14th edn. Baltimore: Williams and Wilkins 2000;2195-245. Pelletier JP, McCollum R, Cloutier JM, et al. Synthesis of metalloproteases and interleukin 6 (1L6) in human osteoarthritic synovial membrane is an IL-l mediated process. J Rheumatol 1995;22: 109-14. Pelletier JP, Mineau F, Ranger P, et al. The increased synthesis of inducible nitric oxide inhibits IL-lra synthesis by human articular chondrocytes: possible role in osteoarthritic cartilage degradation. Ostearthritis Cartilage 1996;4277-84. 103 Pervin A, al-Hakim A, Linhardt RJ. Separation of glycosaminoglycan-dcrived oligosaccharides by capillary electrophoresis using reverse polarity. Anal Biochem 1994;221:182-8. Piperno M, Reboul P, Hellio Le Graverand MP, Peschard MJ. Glucosamine sulfate modulates dysregulated activities of human osteoarthritic chondrocytes in vitro. Ostearthritis Cartilage 2000;8:207-12. Platt D. Articular cartilage homeostasis and the role of growth factors and cytokines in regulating matrix composition. In: Joint Disease in the Horse, McIlwraith CW and Trotter GW, eds. WB Saunders, Philadelphia 1996;29-40 Platt D. The role of oral disease-modifying agents glucosamine and chondroitin sulphate in the management of equine degenerative joint disease. Equine Vet Educ 2001;262-72. Platt D, Bayliss MT. An investigation of the proteoglycan metabolism of mature equine articular cartilage and its regulation by interleukin-l. Equine Vet J 1994;26:297-303. Platt D, Bird J L, Bayliss MT. Ageing of equine articular cartilage: structure and composition of aggrecan and decorin. Equine VetJ 1998;30:43-52. Pool RR, Meagher DM. Pathologic findings and pathogenesis of racetrack injuries. Vet Clin North Am Eq Pract 1990;6zl-30. Poole AR. Proteoglycans in health and disease: Structures and Functions. Biochem J 1986;236:1-14. Poole AR, Webber C, Pidoux I et al. Localization of a dermatan sulfate proteoglycan (DS-PGII) in cartilage and the presence of an immunologically related species in other tissues. J Histol Cytochem 1986;34:619-25. Pouwels MJ, Jacobs JR, Span PN, et al. Short-term glucosamine infusion does not affect insulin sensitivity in humans. J Clin Endocrinol Metab 2001;86:2099-103. 104 Punzi L. The complexity of the mechanisms of action of hyaluronan in joint diseases. Clin Exp Rheumatol 2001;19:242-6. Qian Y, Commane M, Ninomiya-Tsuji J, et al. IRAK-mediated translocation of TRAF6 and TABZ in the interleukin-l-induced activation of NFKB. J Biol Chem 2001:276;41661-7. Radons J, Gabler S, Wesche H et al. Identification of essential regions in the cytoplasmic tail of interleukin-1 receptor accessory protein critical for interleukin-1 signaling. J Biol Chem 2002;277: 16456-63. Reboul P, Pelletier JP, Tardif G et al. The new collagenase, collagenase 3, is expressed and synthesized by human chondrocytes but not by synovial fibroblasts: a role in osteoarthritis. J Clin Invest 1996;97:2011-9. Reid LR, Lowe C, Cornish J, et al. Leukemia inhibitory factor: a novel bone-active cytokine. Endocrin 1990;126: 1416-20. Reginster JY, Deroisy R, Rovatti LC, et al. Long—term effects of glucosamine sulphate on osteoarthritis progression: a randomized, placebo-controlled clinical trial. Lancet 2001;357:251-6. Richardson D, Dodge GR. Effects of interleukin-10 and tumor necrosis factor-a on expression of matrix-related genes by cultured equine articular chondrocytes. Am J Vet Res 2000;61:624-30. Richardson D, Dodge GR. Dose-dependent effects of corticosteroids on the expression of matrix-related genes in normal and cytokine-treated articular chondrocytes. Inflamm Res 2003;52:39-49. Richy F, Bruyere O, Ethgen O et al. Structural and symptomatic efficacy of glucosamine and chondroitin in knee osteoarthritis: A comprehensive meta-analysis. Arch Intern Med 2003;163:1514-22. Rindone JP, Hiller D, Collacott E, et al. Randomized controlled trial of glucosamine for treating osteoarthritis of the knee. West J Med 2000;172:91-4. 105 Roden L. Effect of hexosamines on the synthesis of chondroitin sulphuric acid in vitro. Arkiv Kemi 1956;10:345-52. Rogachefsky RA, Dean DD, Howell DS, et al. Treatment of canine osteoarthritis with insulin-like growth factor-1 (IGF-I) and sodium pentosan polysulfate. Ostearthritis Cartilage 1993 ;1 : 1 05-14. Rohde C, Anderson DE, Bertone AL, et al. Effects of phenylbutazone on bone activity and formation in horses. Am J Vet Res 2000;61:537-43. Ronca F, Palmieri L, Panicucci P, et al. Anti-inflammatory activity of chondroitin sulfate. Ostearthritis Cartilage 1998 (Suppl 6A);14-21. Rossdale PD, Hopes R, Wingfield Digby NJ, et al. Epidemiological study of wastage among racehorses 1982 and 1983. Vet Rec 1985;116:66-69. Saari H, Konttinen VT, Tulamo RN. Concentration and degree of polymerization of hyaluronate in equine synovial fluid. Am J Vet Res 1989:50:2060-3. Sadouk MB, Pelletier JP, Tardif G et al. Human synovial fibroblasts coexpress IL-1 receptor type I and II mRNA. The increased level of IL-1 receptor in osteoarthritic cells is related to an increased level of the type I receptor. Lab Invest 1995;73:347-55. Sadowski T, Steinmeyer J. Effects of non-steroidal anti-inflammatory drugs and dexamethasone on the activity and expression of matrix metalloproteinase-1, matrix metalloproteinase-3, and tissue inhibitor of metalloproteinase-l by bovine articular chondrocytes. Ostearthritis Cartilage 2001;9z407-15. Sakai S, Onose J, Nakamura H, et al. Pretreatment procedure for the microdeterination of chondroitin sulfate in plasma and urine. Anal Biochem 2002;3 02: 169-74. Sandy JD, Gamett D, Thompson V, et al. Chondrocyte mediated catabolism of aggrecan: aggrecanase dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine. Biochem J 1998;335:59-66 . 106 Sandy JD, Neame PJ, Boynton RE, et al. Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain. J Biol Chem 1991;266:8683-5. Sandy JD, Verscharen C. Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo. Biochem J 2001 ;358:615-26. Scheinman RI, Gualberto A, Jewell CM, et al. Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Mol Cell Biol 1995;15:943-53. ' Shikman AR, Kuhn K, Alaaeddine N, et al. N-Acetylglucosamine prevents Il-IB- mediated activation of human chondrocytes. J Immunol 2001;166:5155-60. Schmidt MB, Schoonvbeck J M, Mow VC et al. The relationship between collagen crosslinking and the tensile properties of articular cartilage. In: Proceedings Orthop Res Soc 1987;33:134. Schwenzer R, Siemienski K, Liptay S et al. The human tumor necrosis factor (TNF) receptor-associated factor 1 gene (TRAF 1) is up-regulated by cytokines of the TNK ligand family and modulates TNF-induced activation of NF-kappaB and c-Jun N- terminal kinase. J Biol Chem 1999;274:19368-74. Scott J E. Proteoglycan-fibrillar collagen interactions. Biochem J 1988;252:313-23. Scroggie DA, Albright A, Harris MD. The effect of gluocsamine-chondroitin supplementation on glycosylated hemoglobin levels in patients with Type 2 diabetes mellitus. Arch Intern Med 2003;163: 1587-90. Setnikar I, Giachetti C, Zanolo G. Absorption, distribution and excretion of radioactivity after a single intravenous or oral administration of 14C glucosamine to the rat. Pharmacatherapeutica 1984;32538-50. Setnikar I, Giacchetti C, Zanolo G. Pharmacokinetics of glucosamine in dog and in man. Arzneim-Forshc Drug Res 1986;36:729-35. 107 Setnikar I, Pacini MA, Revel L. Antiarthritic effects of glucosamine sulfate studied in animal models. Arzneim-Forsch Drug Res 1991;41:542-5. Setnikar I, Palumbo R, Canali S, et al. Pharmacokinetics of glucosamine in man. Arzneim-Forsch Drug Res 1993;43 : 10;] 109-13. Setnikar I, Rovati LC. Absorption, Distribution, Metabolism and Excretion of Glucosamine Sulfate. Arzneim-Forshc Drug Res 2001;51:699-725. Shikhman AR, Brinson DC, Lotz MK. Distinct pathways regulate facilitated glucose transport in human articular chondrocytes during anabolic and catabolic responses. Am J Physiol Endocrinol Metab 2004;2862E980-5. Shikhman AR, Kuhn K, Alaaeddine N, et al. N-Acetylglucosamine prevents IL-IB- mediated activation of human chondrocytes.J1mmunol 2001;166:5155-60. Shlopov BV, Lie WR, Mainardi CL, et al. Osteoarthritic lesions: involvement of three different collagenases. Arthritis Rheum 1997;40:2065-2074. Smith MM, Ghosh P. The synthesis of hyaluronic acid by human synovial fibroblasts is influenced by the nature of the hyaluronate in the extracellular environment. Rheumatol Int 1987;7:1 13-22. Spreng D, Sigrist N, J ungi T, et al. Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture. Am J Vet Res 2000;61:530-6. Stove J, Schoniger R, Huch K, et al. Effects of dexamethasone on proteoglycan content and gene expression of IL-lbeta-stimulated osteoarthritis chondrocytes in vitro. Acta Orthop Scand 2002;73 :562-7. Strand E, Martin GS, Crawford MP, et a]. Intra-articular pressure, elastance and range of motion in healthy and injured racehorse metacarpophalangeal joints. Equine VetJ 1999;30:520-7. 108 Su S, Grover J, Roughley PJ, DiBattista JA, et al. Expression of tissue inhibitor of metalloproteinases (TIMP) gene family in normal and osteoarthritis joints. Rheumatol Int 1999;18:183-91. Suzuki K, Enghild JJ, Mordomi T, et al. Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromeolysin). Biochem 1990;29: 10261-70. Tada K, Okazaki T, Sakon S, et al. Critical roles of TRAF2 and TRAFS in tumor necrosis factor-induced NF-KB activation and protection from cell death. J Biol Chem 2001;276: 36530-4. Tak PP, Firestein GS. NF-KB: a key role in inflammatory disease. J Clin Invest 2001;107:7-11. Takaesu G, Surabhi RM, Park K, et al. TAKl is critical for IKB Kinase-mediated activation of the NF -KB Pathway. J Mol Biol 2003:326:105-15. Takeuchi M, Rothe M, Goeddel DV. Anatomy of TRAF2: Distinct domains for nuclear factor-KB activation and association with tumor necrosis factor signaling proteins. J Biol Chem 1996;271:19935- 42. Takeuchi O, Akira S. MyD88 as a bottle neck in Toll/IL signaling. Current Top Microbiol Immunol 2002;270:155-67. Takafuji VA, McIlwraith CW, Howard RD. Effects of equine recombinant interleukin-la and interleukin-III on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants. Am J Vet Res 2002;63:551-6. Takahashi K, Goomer RS, Harwood F et al. The effects of hyaluronan on matrix metalloproteinase-3 (MMP 3), interleukin-lbeta (IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP 1) gene expression during the development of osteoarthritis. Ostearthritis Cartilage 1999;7:182-90. Takahashi K, Hashimoto S, Kubo T, et al. Effect of hyaluronan on chondrocyte apoptosis and nitric oxide production in experimentally induced osteoarthritis. J Rheumatol 2000;27: 1713-20. 109 Takizawa M, Ohuchi E, Yamanaka H et al. Production of tissue inhibitor of metalloproteinase 3 is selectively enhanced by calcium pentosan polysulfate in human rheumatoid synovial synovial fibroblasts. Arthritis Rheum 2000;43:812-20. Tardif G, Pelletier JP, Dupuis M, Hambor J E, Martel-Pelletier J. Cloning, sequencing and characterization of the 5’-flanking region of the human collagenase- 3 gene. Biochem J 1997;323:13-6. Tesoriere G, Dones F, Magistro D et al. Intestinal absorption of glucosamine and N- acetylglucosamine. Experientia 1972;28 :770-1 . Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human articular cartilage: Associations with degenerative changes. Arth Rheum 2001;44:585-94. Thie NM, Prasad NG, Major PW. Evaluation of glucosamine sulfate compared to ibuprofen for the treatment of temporomandibular joint osteoarthritis: a randomized double blind controlled 3 month clinical trial. J Rheumatol 2001;28:1347-55. Thomas B, Thirion S, Humbert L et al. Differentiation regulates interleukin-lbeta- induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen- activated protein kinase. Biochem J 2002;362:367-73. Tobetto K, Yasui T, Ando T, et al. Inhibitory effects of hyaluronan on [14C] arachidonic acid release from labeled human synovial fibroblasts. Jpn J Pharmacol 1992;60:79-84. Todhunter RJ. Anatomy and physiology of synovial joints. In: Joint Disease in the Horse, McIlwraith CW, Trotter GW, eds. WB Saunders, Philadelphia 1996;1-28. Todhunter RJ, Lust G. Polysulfated glycosaminoglycan in the treatment of osteoarthritis. J Am Vet Med Assoc 1994 ;204: 1245-51. Todhunter RJ, Minor RR, Wootton JA et al. Effects of exercise and polysulfated glycosaminoglycan on repair of articular cartilage defects in the equine carpus. J Orthop Res 1993;1 1:782-95. 110 Todhunter RJ, Wootton JA, Lust G et al. Structure of equine type I and type II collagens. Am J Vet Res 1994;38:425-31. Towheed TE, Anastassiades TP, Shea B, et al. Glucosamine therapy for treating osteoarthritis (Cochrane review). Cochrane Database Syst Rev 2001;] :CD002946. Trotter GW. Intra-articular corticosteroids. In: Joint Disease in the Horse, McIlwraith CW, Trotter GW, eds. WB Saunders, Philadelphia 1996;237-253. Trotter GW, Yovich JV, McIlwraith CW, et al. Effects of intramuscular polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage. Can J Vet Res 1989;53:224-30. Trumble TN, Trotter GW, Thom JR, et al. Synovial fluid gelatinase concentrations and matrix metalloproteinase and cytokine expression in naturally occurring joint disase in horses. Am J Vet Res 2001 ;9: 1467-77. Tung J T, Arnold CE, Alexander LH, et al. Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-10 stimulated MMPl, 3 and 13 and TIMP expression in equine chondrocyte cultures. Am J Vet Res 2002;63 :987-93. Tung JT, Fenton J 1, Arnold C, Alexander L, et al. Recombinant equine interleukin- IB induces putative mediators of articular cartilage degradation in equine chondrocytes. Can J Vet Res 2002;66:19-25. Tung J T, Venta PJ, Caron JP. Inducible nitric oxide expression in equine articular chondrocytes: effects of anti-inflammatory compounds. Ostearthritis Cartilage 2002:10;5-12. Tung J T, Venta PJ, Eberhart SW et al. Effects of anti-arthritis preparations on gene expression and enzyme activity of cyclooxygenase-2 in cultured equine chondrocytes. Am J Vet Res 2002;63 :1 134-9. Uebelhart D, Malaise M, Marcolongo R, et al. Intermittent treatment of knee osteoarthritis with oral chondroitin sulfate: a one-year, randomized, double-blind, multi-center study versus placebo. Ostearthritis Cartilage 2004;12:269-76. 11] Uebelhart D, Thonar EJ, Delmas PD, et al. Effects of oral CS on the progression of knee osteoarthritis: a pilot study. Ostearthritis Cartilage 1998;6 (Suppl A) 37-8. Uebelhart D, Thonar EJ, Zhang J, et al. Protective effect of exogenous chondroitin- 4,6-Sulfate in the acute degradation of articular cartilage in the rabbit. Ostearthritis Cartilage 1998;6 (Suppl A):6-13. Uldry M, Ibberson M, Hosokawa M. GLUT2 is a high affinity glucosamine transporter. F EBS Lett 2002;524:199-203. Valhmu WB, Palmer GD, Rivers PA et al. Structure of the human aggrecan gene: exon-intron organization and association with the protein domains. Biochem J 1995;309:535-42. van der Rest M, Mayne R. Type IX collagen proteoglycan from cartilage is covalently cross-linked to type II collagen. J Biol Chem 1988;263:1615-8. Verbruggen G, Goemaere S, Veys EM. Chondroitin sulfate: S/DMOAD (structure/disease modifying anti-osteoarthritis drug) in the treatment of finger joint OA. Ostearthritis Cartilage 1998;6 (Suppl A):37-8. Villiger PM, Geng Y, Lotz M. Induction of cytokine expression by leukemia inhibitory factor. J Clin Invest 1993;91:1575-81. Vincenti MP, Brinckerhoff CE. Early response genes induced in chondrocytes stimulated with the inflammatory cytokine interleukin-113. Arthritis Res 2001;3:381- 8. Vincenti MP, Brinckerhoff CE. Transcriptional regulation of collagenase (MMP 1, MMP 13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis Res 2002;4:157-64. Vincenti MP, Coon CI, Brinckerhoff CE. Nuclear factor kappaB/p50 activates an element in the distal matrix metalloproteinase 1 promoter in interleukin-lbeta- stimulated synovial fibroblasts. Arthritis Rheum 1998:41;1987-94. 112 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function and biochemistry. Circ Res 2003;92:827-39. Volpi N. Electrophoresis separation of glycosaminoglycans on nitrocellulose membranes. Anal Biochem 1996;240:1 14-8. Volpi N. Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by High—Performance Liquid Chromatography and fluorimetric detection with dansylhydrazine. Anal Biochem 2000;277:19-24. Volpi N. Oral bioavailability of chondroitin sulfate (Condrosurf®) and its constituents in healthy male volunteers. Ostearthritis Cartilage 2002;10:768-77. Volpi N. Oral absorption and bioavailability of ichthyic origin chondroitin sulfate in healthy male volunteers. Ostearthritis Cartilage 2003;11:433-41. von Rechenberg B, McIlwraith CW, Akens MK, F et al. Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints. Equine VetJ2000;32:140-50. Wells T, Davidson C, Morgelin M, Bird JL et al. Age-related changes in the composition, the molecular stoichiometry and the stability of proteoglycan aggregates extracted from human articular cartilage. Biochem J 2003;370:69-79. Westling J, Fosang A, Last K, Thompson V et al. ADAMSTS4 cleaves at the aggrecanase site (Glum-Alam) and secondarily at the matrix metalloproteinase site (Asn34l-Phe342) in the aggrecan interglobular domain. J Biol Chem 2002;277:16059- 66. White GW, Stites T, Wynn Jones E et al. Efficacy of intramuscular chondroitin sulfate and compounded acetyl-d-glucosamine in a positive controlled study of equine carpitis. J Equine Vet Sci 2003 ;23 2295-300. White GW, Jones EW, Hamm J, et al. The efficacy of orally administered sulfated glycosaminoglycan in chemically induced equine synovitis and degenerative joint disease. J Equine Vet Sci 1994;14:350-3. 113 Windhaber RA, Wilkins RJ, Meredith D. Functional characterisation of glucose transport in bovine articular chondrocytes. Pflugers Arch 2003;446:572-7. Wu W, Billinghurst RC, Pidoux I et al. Sites of collagenase cleavage and denaturation of type II collagen in aging and osteoarthritic articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 and matrix metalloproteinase 13. Arthritis Rheum 2002;46:2087-94. Yamada H, Kikuchi T, Nemoto O, et al. Effects of indomethacin on the production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 by human articular chondrocytes. J Rheumatol 1996;23:1739-43. Ye H, Park YC, Kreishman M, Wu H. The structural basis for the recognition of diverse receptor sequences by TRAF2. Mol Cell l999;4:321-330. Yin MJ, Yamamoto Y, Gaynor RB.The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature 1998;396:77-80. Yu WH, Yu S, Meng Q, et al. TIMP 3 binds to sulfated glycosaminoglycans of the extracellular matrix. J Biol Chem 2000;275:31226-32. Zafarullah M, Pelletier JP, Cloutier J M, et al. Elevated metalloproteinase and tissue inhibitor of metalloproteinase mRNA in human osteoarthritic synovia. J Rheumatol 1993;20:693-7. 114 CHAPTER 2: DETERMINATION OF A SUBSATURATING DOSE OF RECOMBINANT EQUINE INTERLEUKIN-IB FOR GENES IMPLICATED IN CARTILAGE DEGRADATION IN OSTEOARTHRITIS Summary Objective: To determine a subsaturating dose of recombinant equine interleukin-113 (reIL-113) corresponding to approximately half maximal induction of expression of genes involved in cartilage degradation. Design: Equine chondrocytes in pellet cultures were exposed to incremental doses of reIL-10 at concentrations ranging from 0 to 20,000 pg/ml. RNA was isolated after a 6-hour incubation and effects on gene expression of matrix metalloproteinase 13 (MMP 13), inducble nitric oxide synthase (iNOS), cyclooxygenase 2 (COX 2), aggrecanase-1 (Agg 1), tissue inhibitor of metalloproteinases 1 (TIMP 1), type II collagen, nuclear factor kappaB (N FKB), and interleukin receprot antagonist (ILRa) assessed with quantitative real-time polymerase chain reaction (Q-RT-PCR). Results: reIL-113 resulted in dose dependent saturable up regulation of expression of iNOS, COX 2, Agg 1, MMP 13 and ILRa. The effect on TIMP 1 mRNA was minimal, except at high doses of reIL-113. At low doses (10-500 pg/ml), type II collagen gene expression increased; however, higher doses reduced gene expression. NFKB gene expression was upregulated at all doses except the highest dose (20,000 pg /ml). Conclusion: A subsaturating dose of 500 pg/ml was determined to result in approximately half-maximal effect on the majority of the genes of interest. 115 Introduction The hallmark of osteoarthritis (0A) is the progressive and permanent degeneration of articular cartilage. Establishment of the role of cytokines in OA (Martel-Pelletier et al 1999) prompted the use of cytokines, including interleukins, to create in vitro models of OA. Interleukin-1 (IL-1) is a primary mediator of the degradative processes characteristic of OA, with elevated levels of IL-l-like biological activity in the synovial fluid of horses with clinical OA supportive of its role in the disease (Morris et al 1990, Alwan et al 1991). Pharmacological inhibition of the effects of IL-1 on equine cartilage, as a means to slow or arrest OA progression, illustrates its importance in OA (Caron et al 1996a, F risbie et al 2000, Frean et al 2000). The response of cartilage to IL-1 Stimulation is multifactorial. Extracellular matrix synthesis is inhibited and the production of inflammatory mediators and matrix degrading enzymes predominate, leading to proteoglycan loss, cleavage of type II collagen and degradation of aggrecan, events which are synonymous with 0A. In vitro, IL-1 stimulation induces synthesis of phospholipase A2, COX 2, PGE2, and iNOS; and inhibits collagen synthesis (type 11, IX, and XI) (Cook et al 2000, Murrell et al 1995, Tung et al 2002a). Further, IL-l induces MMP synthesis (Caron et al 1996b, Reboul et al 1996, Richardson et al 2000), GAG release into media and aggrecanase activity (Cawston et a1 1999). Effects on PG homeostasis include both dose dependent induction of PG degradation (Frean et al 2000) and inhibition of PG synthesis (Morris et a1 1994, Frisbie et al 1997, MacDonald et al 1992, Platt et al 1994). 116 Stimulation of cartilage with recombinant human interleukin-1 (rhIL-l) in vitro suppresses synthesis of type II collagen (Goldring et al 1988) and aggrecan (Richardson et al 2000), and induces synthesis of MMPS, PGE2 and NO (Morris et al 1994, Caron et al 1996b), and induces ECM degradation (MacDonald et al 1992, F risbie et al 2000). An age effect was identified, with an overall reduced response to IL-1 in cartilage obtained from older humans and horses (Ismaiel et al 1992, May et al 1992b, Morris et al 1994, MacDonald et al 1992). Further, the response to IL-1 varies not only between species, but also with the source of IL-1, with dissimilar and disproportionate stimulation of PGE2 and MMP synthesis by chondrocytes and synoviocytes following stimulation with rhIL-l and a partially purified form of equine IL-l (May et a1 1990). The form in which chondrocytes are propagated in culture also has an influence on the concentration of cytokine required to produce a given stimulus. Typically, a higher dose is employed for the stimulation of chondrocytes in explants than for monolayer cultures. Recombinant equine interleukin-1 (reIL-1) is a species-specific interleukin developed following concerns that, due to limited sequence identity between equine and human IL-IB, the response of equine cartilage to rhIL-l may not be entirely representative of the in vivo situation (Tung et al 2002a). Human synoviocytes and chondrocytes required only 5% of receptors to be occupied for half maximal stimulation of MMPS by rhIL-IB (Martel-Pelletier et a1 1992). In contrast, 2-3 times 117 greater concentration was needed to saturate all receptor sites to initiate low-level MMP secretion in other species (Chin et al 1990). In equine culture systems, purified reIL-10 resulted in dose saturable up regulation of MMP 1, 3, l3, TIMP], and COX 2 gene expression (Tung et al 2002a); and dose dependently increased PGE2 synthesis and PG release into media (Takafuji et al 2002). While catabolic and anabolic effects were in agreement with previous studies utilizing rhIL-l (MacDonald et al 1992, Platt et al 1994, Morris et al 1994), concentrations of reIL-1 were 40-100 times lower. The choice of an appropriate in vitro model of equine 0A that appropriately reflects the in vivo disease process has been debatable. Both IL-1 and lipopolysaccharide (LPS) have been used in vitro to stimulate OA, and result in similar metabolic responses and cartilage degradation (Fenton et al 2000). However, horses are particularly sensitive to the effects of LPS relative to other species (MacDonald et al 1994). The use of a Species-specific interleukin, at a subsaturating dose, appears to be a useful means of study of a number of pathophysiological events in equine 0A. The objective of the study reported here was to determine the ideal dose of reIL-113 to result in half-maximal induction of a variety of genes implicated in cartilage degradation in CA using chondrocytes in pellet culture. 118 Materials and Methods Tissue Sources-Pellet Cultures Grossly normal metacarpophalangeal articular cartilage was obtained from horses between 2 and 8 years of age that died or were euthanized for reasons other than joint disease. Chondrocyte isolation and propagation in pellet culture was conducted as previously described with minor modifications (Caron et al 1996b). Breifly, cartilage was dissected from the subchondral bone and incubated in physiologic saline (0.9% NaCl) solution containing penicillin (500 U/ml) and streptomycina (500 mg/ml) (25 °C, 1 hour). Chondrocytes were isolated by sequential digestion with pronaseb (1mg/ml, 1 hour) and collagenasea (0.3mg/ml, 18 hours) as previously described. After digestion, cells were separated by sequential centrifugation (300 x g, 10 minutes, repeated 3 times), washed, and re-suspended in 20ml Dulbecco’s modified Eagle’s medium (DMEM):nutrient mixture F-12 (Ham) (1:1)b. The media was supplemented with insulin-transferrin-sodium selenite supplement° (5 ug/ml insulin, 5 ug/ml transferrin, 5 ng/ml sodium selenite), 50 ug/ml ascorbic acid”, amino acids”, 2 ug/ml lactalbumin hydrolysateb, 5 jig/ml Iinoleic acidb, 40 ng/ml thyroxineb, and 100 U/ml penicillin/streptomycin“. The concentration of amino acids was 50% of those previously reported (Rosselot et al 1992). Cell concentration was determined with a hemocytometer and aliquots of 3 X 106 cells were transferred to 15 ml polypropylene centrifuge tubes in 1 ml supplemented serum free media described above. Following centrifugation (300 X g, 5 minutes), pellets were incubated under standard cell culture conditions (37 °C, 95% RH, 5% 119 CO2). Medium was changed every 3 days. Pellet cultures were maintained in serum free media without supplementation with insulin-transferrin-sodium selenite supplement, linoleic acid, thyroxine and ascorbic acid for 2 days prior to the start of an experiment for equilibration. At the conclusion of experiments pellets were collected for isolation of total RNA. RNA Isolation Total RNA was extracted, using a commercial extraction preparationd and RNA isolation kit‘ and following manufacturer’s instructions with minor modifications (Reno et al 1997). Briefly, 1 ml of this agent was added to each pellet after removal of media, incubated (25°C, 5 min), placed on plate shaker (25°C, 5 min) and transferred to microcentrifuge tubes. Following centrifugation (10,000 X g, 10 min), 200 u] chloroform was added to extract total RNA followed by agitation and a second incubation (25°C, 2 min). The aqueous phase containing RNA was collected after centrifugation (4°C, 12,000 X g, 15 min) and RNA precipitated with an equal volume of 75% ethanol. RNA was then purified further with RNeasy mini spin columns‘. Pellets were re-suspended in RN-ase free water (0.1% diethylpyrocarbonate, supplied) and centrifuged (10,000 x g, 1 min) to elute RNA. RNA was analyzed by electrophoresis through 1% agarose gels containing IOug/ml ethidium bromide in 1 x MOPS (3-(N-morpholino) propanesulfonic acid to validate spectrophotometric determination and RNA integrity. RNA was quantified by UV spectrophotometryf, and adjusted with RN-ase free water to 1 rig/u] solutions. 120 Quantitative Real Time PCR One microgram of each RNA sample was treated with Dnaseg to degrade contaminating Single and double stranded DNA. Treated RNA was converted to single stranded cDNA using Superscript II reverse transcriptaseg as recommended by the manufacturer. F inal cDNA pellets were resuspended in 20 u] of RNase-free water. One microliter of each cDNA template primed each PCR reactionh using Taq DNA polymeraseg. PCR derived cDNA was analyzed by electrophoresis through 1.5% agarose gels in Tris-HCL-acetate-EDTA (TAE) gels containing IOug/ml ethidium bromide. cDNA was quantified by UV spectrophotometry, and adjusted with Rnase-free water to SOng/ul. Reference amplicons were developed using equine cartilage RNA-derived cDNA using specific primers designed by Primer Express software Version 2.0i and were synthesized by a commercial facilityj. Nucleotide sequences used for primer design were obtained from public databases (Genbankk) as full length or partial equine sequences were available for glyceraldehyde phosphate dehydrogenase (GAPDH), inducible nitric oxide synthase (iNOS), matrix metalloproteinase 13 (MMP l3), tissue inhibitor of metalloproteinases-l (TIMP 1), aggrecanase-1 (Agg 1), cyclo- oxygenase 2 (COX 2), type II collagen, and interleukin-1 receptor antagonist (ILRa). When no equine sequence was available (nuclear factor kappaB (NFKB), equine expressed sequence tag (EST) sequences corresponding to the target gene were used based on similarity to the corresponding mouse sequence. BLASTNk searches for all of the primer and amplicon sequences were conducted to ensure gene specificity. 12] Optimal concentrations of each set of primers were determined with a primer matrix (lowest standard deviation with no change in cycle to threshold (Ct)). Primer sequences used for each gene are summarized in Table I. Fifty nanograms of sample cDNA primed each real-time PCR reaction. All analyses were conducted in a PE 7700 DNA sequence detection system]. The cDNA templates were combined with optimal concentrations of primers and SYBR Green PCR dye mixi in a total volume of 50 pl and the amplification conducted as recommended by the manufacturer. The PCR conditions were 2 min at 50°C and 10 min at 95°C followed by 40 cycles of extension at 95°C for 15 sec and 1 min at 60°C. Threshold lines were adjusted to intersect amplification lines in the linear portion of the amplification curves. The software automatically recorded the Ct. Analysis of each sample was performed in duplicate, and a standard deviation of <05 set for control of pipetting error. Dissociation curves were checked for each sample to verify that the correct product was amplified with specific melting temperatures compared to those calculated by Primer Express. For each set of amplification reactions, GAPDH was used as an endogenous control for normalization of RNA loading, cDNA synthesis efficiency, and amplification efficiency. Reference amplicons for each gene and primer sets were functional based on test amplifications using equine cartilage RNA-derived cDNA. The supplemented serum free control was used as a calibrator (ie the fold change for control is 1.0). Replicated data was normalized with GAPDH and the fold change in gene expression relative to serum free control treatment was calculated using the Delta Delta Ct method (Livak eta12001). 122 Stimulation with reIL-1,6 ReIL-IB was purified as described previously (Tung et al 2002). After an 8-12 day period to establish a stable metabolism, reIL-10 was added in increments to achieve final concentrations ranging from 0 to 20,000 pg/ml (0, 10, 50, 100, 500, 1000, 5000, 10000, 20000 pg/ml). Pellets were harvested after a 6-hour incubation and RNA isolated for Q-RT-PCR. The subsaturating dose of reIL-l to be used was determined using tissues of 5 horses. Results Inflammatory mediators Exposure of pellet cultures to graduated concentrations of reIL-Ill resulted in dose dependent saturable up regulation of expression of both iNOS and COX 2 (Figure 1). The dose of cytokine corresponding to approximately half maximal induction of expression of COX 2 and iNOS was approximately 500 pg/ml. This dose resulted in 2 to 5-fold increase in expression of both genes. Extracellular Matrix Proteins The effect on type II collagen gene expression was dose dependent (Figure 2). At low doses (10-50 pg/ml), gene expression was increased, however, higher doses inhibited gene expression. There was large variability in expression levels between horses at intermediate doses. Less variability was apparent with higher doses (5,000 - 20,000 Pg/ml), with down regulation of gene expression at these doses for all horses. A dose 123 of 500 pg/ml was selected as a subsaturating dose, with an intermediary effect on type II collagen gene expression in this model. Transcription Factors The effect of increasing reIL-lB dose on NF KB gene expression was variable (Figure 2). The response appeared to be dose dependent, with increased gene expression at all doses apart from the highest dose (20,000 pg/ml), which consistently reduced NFKB expression in all horses. However, maximal elevation in expression appeared biphasic, occurring at 50 and 10,000 pg/ml. One horse (3 year old Quarterhorse) was particularly sensitive to reIL-10 stimulation in terms of NFKB expression. As for other genes investigated, 500 pg/ml appeared a suitable sub-saturating dose for NFKB. Degradative Enzymes and their inhibitors The response to reIL-1B stimulation for aggrecanase-l, MMP 13, TIMP 1 and ILRa was gene dependent (Figure 3). All horses had reduced MMP 13 expression at low doses (10 to 100 pg/ml). A dose of 500 pg/ml resulted in approximately half-maximal stimulation of MMP 13; however, gene expression did not increase markedly with increasing doses up to 10,000 pg/ml. Gene expression appeared to be dose saturable, with reduced expression at the highest dose. Stimulation with reIL-10 resulted in dose dependent saturable up regulation of Agg 1 gene expression. For all but one horse, a dose of 1000 pg/ml resulted in maximal 124 induction of gene expression. Gene expression subsequently decreased at higher doses. Thus, although a dose of 500 pg/ml resulted in maximal gene expression in one of the 5 horses used, 500 pg/ml proved a suitable dose. ReIL-IB stimulation had little effect on TIMP 1 gene expression, except at the highest dose. Although response at this dose was variable between horses, 3 of 5 horses had maximal induction of TIMP 1 expression at this dose. The dose of reIL- IB corresponding to maximal IL-lRa expression varied between horses, with maximal expression between 10 to 500 pg/ml. Doses higher than 500 pg/ml subsequently reduced IL-lRa expression relative to the maximal dose. Discussion Exposure of chondrocytes to reIL-113 resulted in upregulation of gene expression of inflammatory mediators, COX 2 and iNOS, and degradative enzymes, MMP 13 and Agg 1. This IL-l induction of gene expression was in agreement with previous studies utilizing both rhIL-l (Caron et al 1996, Morris et al 1994, Richardson et al 2000) and reIL-1 (Tung et al 2002a), with expression of these genes being both dose dependent and saturable. Minimal effect on TIMP 1 expression was apparent, except at the highest dose of reIL-10. While this is in agreement with some studies in which IL-l had no effect on TIMP 1 mRNA levels (Martel-Pelletier et al 1991), other studies have documented 125 an increase in TIMP expression with exposure to IL (Shingu et a1 1993), including reIL-113 (Tung et al 20023). IL-l stimulation dose-dependently increased IL-lRa gene expression; however this effect appeared to be saturable, with reduced expression at higher doses. IL-l induced IL-lRa synthesis in human articular chondrocytes, and 0A cartilage spontaneously produced higher levels of IL-lRa than normal cartilage (Maneiro et al 2001). A relative deficit in IL-lRa production has been demonstrated in OA synovium (Pelletier et al 1996). IL-lRa functions as a competitive inhibitor of IL-1 R, and is capable of inhibiting a number of pathological events in OA including synoviocyte PGE2 synthesis, chondrocyte collagenase production, and degradation of ECM (Pelletier et al 1999). Elevated IL-lRa expression at low doses in this study may be an attempt by cartilage to protect against and inhibit the effect of IL-1. Higher doses decreased expression, to levels equivalent to or slightly greater than unstimulated controls. Other factors may influence ILRa expression, as nitric oxide has been shown to reduce ILRa expression by chondrocytes stimulated with rhIL-IB (Pelletier et al 1996). The effect of IL-1 stimulation on NF KB expression in equine cartilage has not been previously documented. NFKB is a transcription factor in the IL-1 signaling pathway. NFKB binding Sites are present on the promoter regions of a number of inflammatory genes, including IL-l, MMPS, iNOS, and COX 2 (Mengshol et al 2000, Tak et al 2001). IL-l induced activation of NFKB in rat articular chondrocytes 126 (Gouze et al 2002), and induced a time dependent increase in NFKB activity in human OA cartilage in vitro (Largo et al 2003). In this model, reIL-113 induced NFKB gene expression at all doses except the highest dose. This effect was variable depending on the dose, hence dose related kinetics were not obvious and require further elucidation. Further, variability in response between different horses was high. However, elevation in NFKB expression concurrent with elevation in inflammatory mediators and degradative enzymes such as iNOS, COX 2 and MMP13, may be one mechanism by which IL-l exerts its effects in articular cartilage. The response of type II collagen gene expression was dose dependent. Increased expression at low doses may be representative of an attempt of cartilage at repair, as occurs with the initial stages of OA (Aigner et al 2002). Higher doses reduced type II collagen expression, in agreement with previous studies (Cook et al 2001, Goldring et al 1988), consistent with extracellular matrix degradation as has been previously reported (Tyler et al 1988, Richardson et a1 1997 and 2000). The response of individual horses to IL-1 stimulation varied, in agreement with previous studies in which cartilage derived from some humans was susceptible, and others refractory to IL-1 stimulation (Ismaiel et a1 1992). Further, an age related response to IL-1 stimulation has been documented previously (MacDonald et al 1992). Age may have had an influence in our study because of the diversity in the ages of horses from which cartilage was obtained; however, 3 of the 5 horses used 127 were the same age. The subsaturating dose of reIL-113 determined in this study (500 pg/ml) is lower than that used in other studies utilizing rhIL-IB (Caron et al 1996b, Fenton et al 2000) and reIL-10 (Tung et al 2002a,b). In these studies, doses between 1 and 100 ng/ml were tested, with dose saturable kinetics demonstrated for MMP 1, 3, 13, TIMP 1, COX 2 and iNOS. However, in a further study with monolayer cultures, a subsaturating dose of reIL-10 of 50 pg/ml resulted in half-maximal stimulation of MMP 1 (Tung et al 20020). In that study, the 50 pg/ml dose gave approximately 2-fold increase in gene expression of MMP 3, l3 and TIMP 1. In the study presented here, the equivalent dose failed to upregulate MMP13, Agg 1 and COX 2 but did upregulate iNOS, ILRa and NFKB. Equivalent or higher doses of IL-1 that consistently saturated gene expression in this study have been previously used to determine both response to IL-1 stimulation and the ability of compounds to attenuate the response to IL-1 (Fenton et al 2002). Such studies have been criticized as a detrimental effect on cell viability has been documented in vitro using 20 ng/ml of IL-1 (Dvorak et al 2002, Cook et al 2000). The aim of this study was to determine a dose that may be more representative of the in vivo disease process; however, choice of a subsaturating dose may depend on a number of factors including the species tested, culture conditions, and methodology used to determine gene expression. For instance, expression was quantified with Q- RT-PCR, a method that is more sensitive for determination of mRNA expression in articular cartilage than Northern hybridization (Fehr et al 2000). 128 The development of a subsaturating dose of reIL-1B for genes implicated in cartilage degradation in OA provides a model that may be more representative of the in vivo disease process. While individual horse and gene variation are apparent, this dose of 500pg/ml is thought to be intermediary in its effects on gene expression and will be used in subsequent experiments. 129 <0: 005050505505 05550550555055 ”SS: 53:8 = 25. 0 5555500000505 5050550005503 @830 _ “=25 <5< 55:2 88:03 05555050050< 50055055505055 8:2: a $285: :48 $885 22 < 505 00505050050055 55000553555555 $2.80.. 2 .222 5550 55550555005005 55055005055055 288%. 3:3: 5550 5005505055500 5505050050000 @5802 moz_ 0550 0500555005555 5555055000500 80% :2 :25 5 55000005500550 55000055505050 E :32 N 080 5 0550005505000 5555050005005 $0024. 5 SJ. LOQ—mum owhotrom heath whaEOh flcmmmuuo< Jinn—~00 OEOU deuce“: Ease 03.6833 085-32 03083530 8m 00m: A.mAI.mv 305509. Lon—ta 85>»: can Reich AN 332.3 n «3:. 130 COX2 : 12 .. .9. g 10 -~--— — A *~—~——_~_—~— _ - —--—---—————- a. 8— 5 A i i ————#— __, —-————————— a: l I 0 6-;7-.___#______T ____ .2 E 4 J._ F "i _.-____ _.-_ .___.._ O m c 2 “T”_ O“ ” fl " g o [—1 E] I If] I [3'] I I I 1 ‘il 0 10 50 100 500 1000 5000 10000 20000 Dose reIL-18 (pg/ml) iNOS c 10 .2 a u a 8- — . e i " 3 6 o T.— ‘ i—Ii— H .2 a 4 J____.___.__.____ _ 1- "1 é .. "' _I‘ j F":- c 2 ~—-.—H wI . " l o . s 0 [:I 15 e 0 10 50 100 500 1000 5000 10000 2000 Dose reIL-1B (pglml) Figure 1 (chapter 2): Relative expression of inflammatory mediators, COX 2 and iNOS, in equine chondrocyte cultures in response to graduated doses of recombinant equine interleukin-1 B (reIL-1 B). 131 Type II Collagen § 3 g” __-- _---__L_LL.L_- a w—---- -~+ -w-w—— 0 __,_ *fi _____ ____~ .2 . 5 __ *1, ,-.L-____LL O 0‘ ‘I‘r. .. . _ _.__.. c a E -, .fi, ,1: 100 500 1000 5000 10000 20000 Dose reIL-1B (pglml) NFKB : 8 .2 7-2- _- - m. _L__ _ f _L §6-- i -_ H +_ _ -—_____ a .11 5-—~ —- ——-_2 —— ———— -————fi 3 4 JL_ 7 4— 77 __ fi_ _ 33-” ~5- — ”WIT—~— O m 2.,T_ __ 4b,_ 1' __._._, .- §“T3 “WE—T ‘—@~ “er 5 0 I fl I I I I I I 0 10 50 100 500 1000 5000 10000 20000 Dose reIL-1B (991ml) Figure 2 (chapter 2): Relative expression of extracellular matrix protein, type II collagen, and transcription factor, NFKB, in equine chondrocyte cultures in response to graduated doses of recombinant equine interleukin-1B (reIL-1 B). 132 Agg 1 .5 5 g. _,___ _-_-_--__u.-_- 3 m3 77— ,‘m(_ -~——————~~H 0» _ > I" S 21 -— —- I ~- 2 % c 1 4 b 1' O E 0 I T I —I I 100 500 1000 5000 10000 20000 Dose reIL-1B (pg/ml) MMP 13 3 2.5 -- A — - —— — — ~— 2 ._. A I _ 1.5 «l___. ______ -_ 1- m l I. 0.5 -— ”:1— ,_2_ H o OTB I I I I I H l T 0 10 50 100 500 1000 5000 10000 20000 Dose reIL-1B (pglml) Mean Relative Expression Figure 3A (chapter 2): Relative expression of degradative enzymes MMP 13 and Agg 1 in equine chondrocyte cultures in response to graduated doses of recombinant equine interleukin-1 B (reIL-1 B). 133 TIMP 1 Mean Relative Expression 0 10 50 100 500 1000 5000 10000 20000 Dose reIL-1B (pg/ml) ILRa 14 Mean Relative Expression .. F'I'WI—I—I— *TIIT I 100 500 1000 5000 10000 20000 Dose reIL-1B (pglml) Figure 3B (chapter 2): Relative expression of inhibitors (TIMP 1, ILRa) in equine chondrocyte cultures in response to graduated doses of recombinant equine interleukin-1 B (reIL-1 B). 134 References Aigner T, McKenna L. Molecular pathology and pathobiology of osteoarthritis cartilage. Cell Mol Life Sci 2002;59:5-18. Alwan WH, Carter SD, Dixon JB, et al. Interleukin-1 like activity in synovial fluids and sera of horses with arthritis. Res Vet Sci 1991;51:72-7. Caron JP, Fernandes JC, Martel-Pelletier J, et al. Chondroprotective effects of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-1 expression. Arthritis Rheum 1996;39:1535-44. Caron JP, Tardif G, Martel-Pelletier J, et al. Modulation of matrix metalloprotease l3 (collagenase 3) gene expression in equine chondrocytes by IL-1 and corticosteroids. Am J Vet Res 1996;57:1631-4. Cawston T, Billington C, Cleaver C, et al. The Regulation of MMPS and TIMPS in cartilage turnover. Ann N Y Acad Sci 1999;878:120-9. Chin JE, Horuk R. IL-1 receptor on rabbit articular chondrocyte: relationship between biological activity and receptor binding kinetics. FASEB J l990;4: 1481-7. Cook JL, Anderson C, Kreeger JM, et al. Effects of human recombinant interleukin-1B on canine articular chondrocytes in three-dimensional culture. Am J Vet Res 2000;7z766- 70. Dvorak LD, Cook J L, Kreeger J M, et al. Effects of carprofen and dexamethasone on canine chondrocytes in a three-dimensional culture model of osteoarthritis. Am J Vet Res 2002;63:1363-9. Fehr JE, Trotter GW, Oxford J T, et al. Comparison of Northern blot hybridization and a reverse transcriptase-polymerase chain reaction technique for measurement of mRNA expression of metalloproteinases and matrix components in articular cartilage and synovial membrane from horses with osteoarthritis. Am J Vet Res 2000;61:900-5. Fenton JI, Chlebek-Brown KA, Caron JP, et al. Effect of glucosamine on interleukin-1- conditioned articular cartilage. Equine Vet J 2002 (Suppl);34:219-23. 135 F enton JI, Chlebek-Brown KA, Peters TL, et al. Glucosamine HCl reduced equine articular cartilage degradation in explant culture. Osteoarthritis Cartilage 2000;8:258-65. Frean SP, Lees P. Effects of polysulfated glycosaminoglycan and hyaluronan on prostaglandin E2 production by cultured equine synoviocytes. Am J Vet Res 2000;61:499- 505. Frisbie DD, Nixon AJ. Insulin-like growth factor 1 and corticosteroid modulation of chondrocyte metabolic and mitogenic activities in interleukin l-conditioned equine cartilage. Am J Vet Res 1997;58:524-30. Frisbie DD, Sandler EA, Trotter GW, et al. Metabolic and mitogenic activities of insulin- like growth factor-1 in interleukin-1 conditioned equine cartilage. Am J Vet Res 2000;61:436-41. Goldring MB, Birkhead J, Sandell LJ, et al. Interleukin 1 suppresses expression of cartilage-specific types 11 and IX collagens and increases types I and III collagens in human chondrocytes. J Clin Invest l988;82:2026-37. Gouze JN, Bianchi A, Bécuwe P, et al. Glucosamine modulates IL-l-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-KB pathway. FEBS Letters 200225 1 O;166-70. Ismaiel S, Atkins RM, Pearse MF et al. Susceptibility of normal and arthritic human articular cartilage to degradative stimuli. Br J Rheumatol 1992;31:369-73. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-8. Largo R, Alvarez-Soria MA, Diez-Ortego I, et al. Glucosamine inhibits IL-lB-induced NFKB activation in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 2003:] 1290-8. MacDonald MH, Stover SM, Willits NH, et al. Effect of bacterial lipopolysaccharides on sulfated glycosaminoglycan metabolism and prostaglandin E2 synthesis in equine cartilage explant cultures. Am J Vet Res 1994;55:1127-38. 136 MacDonald MH, Stover SM, Willits NH, et al. Regulation of matrix metabolism in equine cartilage explant cultures by interleukin 1. Am J Vet Res 1992;53:2278-85. Maneiro E, Lopez-Armada MJ, Fernandez-Sueiro J L, et a1. Aceclofenac increases the synthesis of interleukin 1 receptor antagonist and decreases the production of nitric oxide in human articular chondrocytes. J Rheumatol 2001;28:2692-9. Martel-Pelletier J, Alaaeddine N, Pelletier JP. Cytokines and their role in the pathophysiology of osteoarthritis. Front Biosci l999;4:D694-703. Martel-Pelletier J, McCollum R, DiBattista JA, et al. The interleukin-1 receptor in normal and 0A human articular chondrocytes is the type-1 receptor: Binding kinetics and biological function. Arthritis Rheum 1992;35:530-40. Martel-Pelletier J, Zafarullah M, Kodama S, et al. In vitro effects of interleukin 1 on the synthesis of metalloproteases, TIMP, plasminogen activators and inhibitors in human articular cartilage. J Rheumatol Suppl 1991 ;27280-4. May SA, Hooke RE, Lees P. The characterization of equine interleukin-1. Vet Immunol Immunopathol 1990;24:169-75. May SA, Hooke RE, Lees P. Species restrictions demonstrated by the stimulation of equine cells with recombinant human interleukin-l. Vet Immunol Immunopathol 1992;30:373-84. Mengshol JA, Vincenti MP, Coon CI, et al. Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-JUN N- tenninal kinase and nuclear factor KB: Differential regulation of collagenase l and 3. Arthritis Rheum 2000;43:801-1 1. Morris EA, MacDonald BS, Webb AC, et al. Identification of interleukin-1 in equine osteoarthritic joint effusions. Am J Vet Res 1990;51:59-64. Morris EA, Treadwell BV. Effect of interleukin 1 on articular cartilage from young and aged horses and comparison with metabolism of osteoarthritic cartilage. Am J Vet Res 1994;55:138-46. 137 Murrell GA, Jang D, Williams RJ. Nitric oxide activates protease enzymes in articular cartilage. Biochem Biophys Res Commun 1995;206:15-21. Pelletier JP, Lascau-Coman V, Jovanovic D, et al. Selective inhibition of inducible nitric oxide synthase in experimental osteoarthritis is associated with reduction in tissue levels of catabolic factors. J Rheumatol 1999;26:2002-14. Pelletier JP, Mineau F, Ranger P, et al. The increased synthesis of inducible nitric oxide inhibits IL-lra synthesis by human articular chondrocytes: possible role in osteoarthritic cartilage degradation. Osteoarthritis Cartilage 1996;4z77-84. Platt D, Bayliss MT. An investigation of the proteoglycan metabolism of mature equine articular cartilage and its regulation by interleukin-1. Equine Vet J 1994;26:297-303. Reboul P, Pelletier JP, Tardif G et al. The new collagenase, collagenase 3, is expressed and synthesized by human chondrocytes but not by synoial fibroblasts: a role in osteoarthritis. J Clin Invest 1996;97:2011-9. Reno C, Marchuk L, Sciore P et a1. Rapid isolation of total RNA from small samples of hypocellular, dense connective tissues. Biotechniques 1997;22:1082-6. Richardson D, Dodge G. Cloning of equine type II procollagen and the modulation of its expression in cultured equine articular chondrocytes. Matrix Biol 1997;16:59-64. Richardson D, Dodge G. Effects of interleukin-lbeta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes. Am J Vet Res 2000;61:624-30. Rosselot G, Reginato AM, Leach RM. Development of a serum-free system to study the effect of growth hormone and insulinlike grth factor-I on cultured postembryonic growth plate chondrocytes. In Vitro Cell Dev Biol 1992;A:235-44. Shingu M, Nagai Y, Isayama T, et al. The effects of cytokines on metalloproteinase inhibitors (TIMP) and collagenase production by human chondrocytes and TIMP production by synovial cells and endothelial cells. Clin Exp Immunol 1993;94:145-9. 138 Tak PP, Firestein GS. NF-KBZ a key role in inflammatory disease. J Clin Invest 2001;107:7-11. Takafuji VA, McIlwraith CW, Howard RD. Effects of equine recombinant interleukin- la and interleukin-H3 on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants. Am J Vet Res 2002;63:551-6. Tung JT, Fenton J I, Arnold C, et al. Recombinant equine interleukin-1 [3 induces putative mediators of articular cartilage degradation in equine chondrocytes. Can J Vet Res 2002;66:19—25. Tung JT, Venta PJ, Caron JP. Inducible nitric oxide expression in equine articular chondrocytes: effects of anti-inflammatory compounds. Osteoarthritis Cartilage 2002:10;5-12. Tung JT, Arnold CE, Alexander LH, et al. Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-10 stimulated MMP], 3 and 13 and TIMP expression in equine chondrocyte cultures. Am J Vet Res 2002;63:987-93. Tyler JA, Benton HP. Synthesis of type II collagen is decreased in cartilage cultured with interleukin 1 while the rate of intracellular degradation remains unchanged. Coll Relat Res 1988;82393-405. 139 CHAPTER 3: GLUCOSAMINE AND CHONDROITIN SULFATE REGULATION OF MEDIATORS OF OSTEOARTHRITIS IN RECOMBINANT EQUINE INTERLEUKIN-IB STIMULATED EQUINE CHONDROCYTES IN PELLET CULTURE. Summary Objective: To determine if glucosamine (GLN) or chondroitin sulfate (CS), at concentrations approximating those achieved by oral administration, influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes. Methods: Using equine chondrocytes in pellet culture stimulated with a sub-saturating dose of recombinant equine interleukin - 1B, the effects of preincubation with GLN (2.5 - 10.0 ug/ml) and CS (5.0 - 50.0 pg/ml) on gene expression of matrix metalloproteinases (MMPS) 1,2,3,9,13, aggrecanase (Agg) 1,2, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX 2), nuclear factor KB (N F KB) and cjun-N-terminal kinase (JNK) were assessed with quantitative real-time polymerase chain reaction (Q-RT-PCR). Results: Glucosamine significantly reduced reIL-10 induced mRNA expression of MMP 13, Agg 1, and JN K at 10.0 ug/ml. A trend for reduction in cytokine-induced expression was also observed for iNOS (P = 0.06) and COX 2 (P = 0.08). Chondroitin sulfate had no effect on gene expression at the concentrations tested. Conclusion: Concentrations of GS achieved following oral administration in horses exerts pre-translational regulation of some putative mediators of osteoarthritis, an effect that may contribute to the cartilage-sparing properties of this aminomonosaccharide. 140 Based on this study, the influence of CS on pre-translational regulation of these selected genes is limited. Introduction Osteoarthritis (OA) remains an important and expensive cause of lameness in affected horses. Although a variety of factors can initiate the disease process, ultimately all articular tissues are affected. The hallmark of 0A is the degeneration of the articular cartilage matrix, attributed to an excess production of proinflammatory cytokines (Martel-Pelletier et al 1999). Interleukin-1 B (IL-113) is widely accepted as one of the cytokines that plays a pivotal role in the pathophysiology of OA (Pelletier et al 1993, Tung et a1 2002). This cytokine induces a number of catabolic events in both synoviocytes and chondrocytes, including induction of genes of matrix degrading proteinases such as the metalloproteinases (MMPS) and aggrecanases, as well as a number of other inflammatory mediators including inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX 2) (Tung et al 2002, Cook et al 2000, Morris et al 1994, MacDonald et al 1992, Richardson et a1 2000), Accumulating evidence indicates that many of the effects of IL-lB on mediators of osteoarthritis pathophysiology are effected through the activation of transcription factors such as nuclear factor kappa B (NFKB) and activator protein —1 (AP-1) (Mengshol et al 2000, Vincenti et al 2002). Oral administration of nutraceuticals containing glucosamine or chondroitin sulfate or both has enjoyed considerable vogue as a symptomatic treatment of DA in man and 141 domestic animals. On-going research in this area indicates that these nutraceuticals may possess chondroprotective or cartilage-sparing properties. For example, a chondroprotective effect has been supported by two studies indicating that glucosamine prevented knee joint space narrowing in OA patients over a three-year period (Pavelka et al 2002, Reginster et al 2001). Similarly, in a randomized, double-blind, placebo- controlled trial, chondroitin sulfate was found to be protective against radiographic progression of finger joint OA (Verbruggen et al 1998). Certain effects of these substances on cellular metabolism have received recent attention. Glucosamine acts as a substrate for and enhances the production of proteoglycans and glycosaminoglycans (Bassleer et al 1998b). Further, glucosamine prevents the repression of galactose B-l,3-glucuronosyltransferase I, a key biosynthetic enzyme in glycosaminoglycan synthesis (Gouze et al 2001). Glucosamine inhibits proteoglycan loss, prostaglandin production, iNOS, and MMP activity in equine cartilage explants stimulated with LPS or human and equine recombinant interleukin-1 (F enton et al 2000a, b, Orth et al 2002, Byron et al 2003). Although purported to have favorable effects on chondrocyte metabolism, chondroitin sulfate has been less well characterized than glucosamine with respect to its anti-catabolic functions. In an in vitro culture system using human chondrocytes in clusters, chondroitin sulfate was protective against IL-l induced deleterious effects on sulfated proteoglycan, and type II collagen synthesis (Bassleer et al 1998a). Chondroitin sulfate stimulates hyaluronic acid production by synoviocytes (N ishikawa et al 1985). 142 Recent reports suggest that at least some of the anti-catabolic effects of glucosamine are exerted at the pre-translational level. Modulation of cytokine and endotoxin-stirnulated induction of MMPS and aggrecanases in chondrocytes has been demonstrated in vitro (Byron et al 2003, Sandy et al 1998, Dodge et al 2003). Specifically, glucosamine was shown to reduce expression of MMP 1, MMP 3, and MMP 13 in monolayer cultures of equine chondrocytes (Byron et al 2003) and MMP 3 in both rat chondrocytes (Gouze et al 2001) and human osteoarthritic chondrocytes (Dodge et a1 2003). Recent data suggest that inhibition of IL-1 induced synthesis of inflammatory mediators and matrix degrading proteinases by glucosamine may occur via reducing the activity of certain cell signaling pathways. For example, the transcription factors NFKB and AP-l are increased in OA cartilage and IL-1 induced NFKB and AP-l activity is associated with enhanced transcription of the aforementioned mediators of cartilage degradation (Vincenti et a1 2001, Mengshol et al 2001). Glucosamine inhibits NFKB binding in a dose dependent manner as well as preventing IL-l induced translocation of p50 and p65 subunits of NFKB to the nucleus in osteoarthritic human cartilage (Largo et al 2003). To date, most in vitro research in this area has been performed with concentrations of GLN and CS that exceed those obtained by oral administration (Fenton et al 2000a, b, Orth et al 2002, Byron et al 2003, Sandy et a1 1998, Largo et al 2003, Goouze et al 2002, F enton et al 2002). Depending on the species and molecular weight of CS, concentrations of CS in serum after oral administration range from 19 to 208 ug/ml, with a cumulative effect afier multiple dosing (Adebowale et al 2002, Du et al 2002, 2004). Glucosamine concentrations after oral dosing are in the 1.25 to 20 ug/ml range, with concentrations up 143 to 350 ;1ng possible after intravenous administration (Liang et al 1999, Adebowale et al 2002, Du et al 2002, 2004). Thus, the objective of the study reported here was to determine the effect concentrations of GLN (2.5 — 10.0 ug/ml) and CS (5.0-50.0 ug/ml) that more closely approximate those achieved following oral administration have on gene expression of a number of mediators of cartilage catabolism in OA using equine chondrocytes in pellet culture stimulated with recombinant equine IL-1 [3 (reIL—1B). Materials and Methods Tissue Sources- Pellet Culture Grossly normal metacarpophalangeal articular cartilage was obtained from horses between 2 and 8 years of age that died or were euthanized for reasons other than joint disease. Chondrocyte isolation and propagation in pellet culture was conducted as previously described with minor modifications (Byron et al 2003, Caron et al 1996). Briefly, cartilage was dissected from the subchondral bone and following incubation in penicillin (500 U/ml) and streptomycina (500 mg/ml) (25 °C, 1 hour), chondrocytes were isolated by sequential digestion with pronaseb (lmg/ml, 1 hour) and collagenase“ (0.3mg/ml, 18 hours). After digestion, the cells were separated by sequential centrifugation (300 x g, 10 minutes, repeated 3 times), washed, and re-suspended in 20ml Dulbecco’s modified Eagle’s medium (DMEM):nutrient mixture F-12 (Ham) (1:1)”. The media was supplemented with insulin-transferrin-sodium selenite supplementc (5 ug/ml insulin, 5 ug/ml transferrin, 5 ng/ml sodium selenite), 50ug/ml ascorbic acidb, amino acids”, 2 pg/ml lactalbumin hydroslyateb, 5 pg/ml linoleic acid”, 40 ng/ml thyroxine”, and 144 100 U/ml penicillin/streptomycin“. The concentration of amino acids was 50% of those previously reported (Rosselot et al 1992). Cell concentration was determined with a hemocytometer and aliquots of 6 X 106 cells were transferred to 15 ml polypropylene centrifuge tubes in 1 ml supplemented serum free media described above. Following centrifugation (300 X g, 5 minutes), pellets were incubated under standard cell culture conditions (37°C, 95% RH, 5% C02). Medium was exchanged every 3 days. Pellet cultures were maintained in serum free media without supplementation with insulin-transferrin-sodium selenite supplement, linoleic acid, thyroxine, and ascorbic acid for 2 days prior to the start of an experiment for equilibration. At the conclusion of experiments pellets were collected for isolation of total RNA. RNA Isolation Total RNA was extracted, using a commercial extraction preparationd and RNA isolation kit“ and following manufacturer’s instructions with minor modifications (Reno et al 1997) Briefly, 1 ml of this agent was added to each pellet after removal of media, incubated (25°C, 5 minutes), placed on a plate shaker (25°C, 5 minutes) and transferred to microcentrifuge tubes. Following centrifugation (10,000 X g, 10 minutes), 200 pl chloroform was added to extract total RNA followed by agitation and a second incubation (25°C, 2 minutes). The aqueous phase containing RNA was collected after centrifugation (4°C, 12,000 X g, 15 minutes) and RNA precipitated with an equal volume of 75% ethanol. RNA was then purified further with RNeasy mini spin columns‘. Pellets 145 were re-suspended in RN-ase free water (0.1% diethylpyrocarbonate, supplied) and centrifuged (10,000 x g, 1 minute) to elute RNA. RNA was analyzed by electrophoresis through 1% agarose gels containing 10ug/ml ethidium bromide in 1 x MOPS (3-(N- morpholino) propanesulfonic acid) to validate spectrophotometric determination and RNA integrity. RNA was quantified by UV spectrophotometryf, and adjusted with RN- ase free water to 1 ug/ul solutions. Quantitative Real Time PCR Two micrograms of each RNA sample was treated with DNase I8 to degrade contaminating single and double stranded DNA. Treated RNA was converted to single stranded cDNA using Superscript II reverse transcriptase8 as recommended by the manufacturer. cDNA was quantified by UV spectrophotometry, and adjusted with Rnase-free water to 25ng/ul. Reference amplicons were developed using specific primers designed by Primer Express softwareh and synthesized by commercial facilitiesi (Tables I and II) Nucleotide sequences used for primer design were obtained from public databases (Genbankj) as full or partial length equine sequences were available for 18s ribosomal subunit (18s), B-actin, Bz-microglobulin, glyceraldehyde phosphate dehydrogenase (GAPDH), inducible nitric oxide synthase (iNOS), matrix metalloproteinases 1,2,3 and 13 (MMP 1, 2, 3, l3), aggrecanases-1 and —2 (Agg 1, 2), cyclooxygenase 2 (COX 2), ribosomal protein L19 (RPL19), and ubiquitin. When no equine sequence was available [cJun N-terminal kinase (JNK), nuclear factor kappaB (NFKB), MMP 9], equine expressed sequence tag (EST) 146 sequences corresponding to the target gene were used, based on similarity to the corresponding bovine, human or mouse sequence. BLASTNJ searches for all of the primer and amplicon sequences were conducted to ensure gene specificity. Optimal concentrations of each set of primers were determined with a primer matrix [lowest standard deviation with no change in cycle to threshold (CT)]. Fifty nanograms of sample cDNA primed each real-time PCR reaction. All analyses were conducted in an ABI PRISM 7700 sequence detection system". The cDNA templates were combined with optimal concentrations of primers and SYBR Green PCR dye mixh in a total volume of 50 ul and the amplification conducted as recommended by the manufacturer. The PCR conditions were 2 min at 50°C and 10min at 95°C followed by 40 cycles of extension at 95°C for 15 sec and 1 min at 60°C. Threshold lines were adjusted to intersect amplification lines in the linear portion of the amplification curves. The software automatically recorded the CT. Analysis of each sample was performed in duplicate, and a standard deviation of <05 between replicates set as a criteria for inclusion of data. Reference amplicons for each gene and primer sets were functional based on test amplifications using equine cartilage RNA-derived cDNA. Replicated data was normalized against the geometric average of 3 endogenous controls (183, GAPDH, Bz-microglobulin) (V andesompele et al 2002), and the fold change in gene expression relative to serum free control was calculated using the 2(-AACT) method (Livak et a1 2001). 147 Determination of a Sub-saturating Dose of reIL-1 ,6 ReIL-lB was purified as described previously (Tung et al 2002). Chondrocyte pellet cultures were established as described above. After an 8-12 day period to establish a stable metabolism, reIL-IB was added in incremental doses to achieve concentrations ranging from 0 to 20,000 pg/ml. Pellets were harvested after a 6-hour incubation and RNA isolated for Q-RT-PCR, with data normalized to GAPDH. Effects on expression were quantified for MMP 13, iNOS, COX 2, and Agg 1. This was repeated using tissue from 5 different horses. Selection of housekeeping genes for use as endogenous controls in subsequent studies Six housekeeping genes (Table I) were selected based on the availability of full or partial length equine sequences for commonly used endogenous controls. Validation of the effect of experimental treatment on the expression of each housekeeping gene was determined for each horse using the 2(-AC’T) method, where AC’T = Cnmmm) — Cuscmm rm comm.) (Livak et a1 2001), with the fold change [2(-AC’T)] expressed as an average fold change from the mean and the standard deviation as the maximum fold change (maximum variability) (Dheda et a1 2004). Absolute CT values were used as an indicator of the level of gene expression, and the overall median expression level (CT) and deviation from median for each housekeeping gene used as an additional measure of gene stability. To determine gene amplification efficiency, cDNA was serially diluted (10-100 ng/ml), and average CT calculated for each gene of interest and each housekeeping gene. The 148 ACT (Cngenc ofimflcst) — CT (housekeeping gem) was determined and plotted as a function of log cDNA dilution. The value of the slope of the regression is a measure of the difference in amplification efficiencies between the gene of interest and the housekeeping gene. A difference in efficiency of <01 was determined for normalization of each gene of interest to a particular housekeeping gene (Livak et al 2001). Selection of the 3 endogenous controls to be used for normalization was based on amplification efficiency, least variability, and level of expression. Effect Of Glucosamine and Chondroitin Sulfate on Gene Expression Pellet cultures were placed in fresh supplemented serum free media described above. After a 5-7 day period to establish a stable metabolism, GLNk (2.5, 5 and 10.0 rig/ml) or CS1 (5.0, 10.0, 20.0 and 50.0 rig/ml) were added to all pellet cultures except positive and negative controls. One hour later, re IL-1 was added at a final concentration of 500 pg/ml to all pellet cultures except negative controls. After a 12-hour incubation under standard cell culture conditions RNA was isolated for Q-RT-PCR. Genes studied included MMP 1, MMP 2, MMP 3, MMP 9, MMP 13, Agg 1, Agg 2, iNOS, COX 2, NFKB, and JN K. This was repeated using tissue from 8 different horses. Statistical Analysis Means for relative expression for each gene of interest included only those horses for which the sub-saturating dose of reIL-113 resulted in a 2-fold or greater up-regulation of expression from resting (control) levels. Distributions were tested for normality using the method of Kolmogorov-Smirnov. When required, means were loglo transformed 149 followed by analysis using a two-way ANOVA (blocked by horse). Post hoc testing was conducted using the Duncan’s multiple range test. A P value < 0.05 was considered significant. A P value < 0.10 was regarded as a statistical trend. Results Determination of a Sub-saturating Dose of reIL-1 ,6 Exposure of pellet cultures to graduated concentrations of reIL-1B resulted in dose dependent saturable induction of expression of Agg 1, MMP 13, iNOS and COX 2 (Figure 1). The dose of cytokine corresponding to approximately half maximal induction of expression of these genes varied from 100-1000 pg/ml. For this reason, the intermediate dose of 500 pg/ml was selected for use in subsequent experiments. This concentration of reIL-18 resulted in approximately 2 to 5-fold increase in expression of the 4 genes. Based on the sizeable variability in data in the subsequent experiments, an analogous dose-response protocol was repeated using cartilage from 4 different horses, with an incubation of 12 hours prior to RNA isolation and amplification with primers for COX 2 with comparable results (Figure 2). Selection of housekeeping genes for use as endogenous controls in subsequent studies Levels of gene expression for available housekeeping genes varied substantially and there were substantial differences in individual housekeeping gene stability between horses. Amplification efficiency varied for each housekeeping gene, with GAPDH the most suitable for comparison for 55% (6/11) of the genes studied. Three housekeeping genes (18s, GAPDH and Bg-microglobulin) were subsequently chosen based on expression 150 level, amplification efficiency, and the lowest level of variability, both overall and for each horse. Effect Of Glucosamine and Chondroitin Sulfate on Gene Expression Glucosamine significantly reduced reIL-113 induced mRNA expression of MMP l3, and Agg 1, at 10.0 jig/ml (Figures 3-4). A trend for reduction in cytokine-induced expression was also observed for iNOS (P = 0.06), COX 2 (P = 0.08) (Figures 5-6). Pellets treated with 10.0 jig/ml of GS had significantly reduced expression of JN K compared to positive (reIL-1B) controls, however, for this gene, the expression of positive and negative controls was comparable. Glucosamine at a concentration of 10 ug/ml reduced IL-l induced stimulation of Agg 2 and NFKB, however the effect was not significant (P = 0.13 and 0.11 respectively). The dose of reIL-IB employed failed to significantly up-regulate MMP 9 expression. Chondroitin sulfate had no significant effect on gene expression at the concentrations tested. Discussion While the potential cartilage-sparing role of glucosamine and chondroitin sulfate has been studied by a variety of means, the specific mechanism(s) of action and the minimally effective concentrations remain to be established. We conducted a Q-RT-PCR based study to characterize the effects on gene expression of GLN and CS that corresponds to concentrations that approximate those achieved by oral administration in monogastrics (Liang et al 1999, Adebowale et al 2002, Du et a1 2002, 2004). Our data support the hypothesis that GLN is capable of pre-translational regulation of reIL-IB-induced 151 stimulation of at least some of the proteins implicated in the process of cartilage degradation at a concentration of 10 rig/ml. Specifically, glucosamine led to a significant reduction of cytokine-stimulated expression of MMP 13 and Agg 1 and a statistical trend (P < 0.1) for iNOS and COX 2. These findings supplement those of previous experiments in our and other laboratories that were conducted with concentrations higher than those found in the plasma of animals administered GLN and CS (Orth et al 2002, Byron et al 2003, Sandy et al 1998, Dodge et al 2003). These observations parallel those of previous publications and provide further evidence supporting a cartilage-sparing effect of this aminomonosaccharide. In contrast to our findings for MMP 13 and Agg 1, while recombinant equine IL-l significantly up-regulated the expression of MMP 1 and MMP 3, and Agg 2 there was no significant influence of GLN treatment. For MMPS l and 3, there was no effect whatsoever. Although not significant, GS at 10ug/ml reduced expression of Agg 2 to levels approximately 25% those of the cytokine control. The lack of an effect of GLN for MMP 1 and 3 is in contrast to previous reports utilizing higher doses of GLN (F enton et al 2000a, Byron et al 2003, Nakamura et al 2004); however, our results parallel another report in which GS at 10 ug/ml failed to significantly repress IL-1 induced MMP production (Nakamura et a1 2004). Differences in responses among the MMPS may be due to experimental design and/or the variability inherent in these particular experiments. For example, pre-incubation of rat chondrocytes with GLN, albeit at a higher dose, down regulated MMP 3 expression (Gouze et a1 2001). The potential for differential regulation of MMPS by GLN should be considered as has been documented in other studies (Orth et 152 al 2002, Dodge et al 2003, Fenton et al 2002). For example, Dodge et al showed that GLN was able to modulate MMP 3 but not MMPl activity (Dodge et al 2003). Another potential reason for this observation is divergence in the regulation of these proteins. In vitro, both temporal and dose dependent differential regulation has been documented (Mengshol et al 2000, Salminen et al 2002, Thompson et al 2001, Koshy et al 2002). This may be firrther complicated by the coordinate regulation of MMPS and aggrecanases (Kevorkian et al 2004) Unlike the other MMPS examined, significant up-regulation of MMPS 2 and 9 by reIL-113 was not observed. This is in keeping with previous studies in chondrocytes, with either no change or mild increases in expression induced by IL-1 (Clegg et al 1999, Sasaki et a1 1996). This may be in part related to basal levels of both MMPS in control samples, as secretion of both MMPs by unstimulated chondrocytes has been suggested (Thompson et al 2001). The gelatinases are proposed to have a secondary role in OA, as collagen degradation occurs only afier the collagenases such as MMP 13 have cleaved the triple helix of collagen (Nagase et a1 1999). The effect on MMP 2 and 9 and may also relate to the differences between normal and 0A chondrocytes, reflecting the differential expression profiles of MMPS in early compared to late OA cartilage (Kevorkian et a1 2004, Aigner et al 2001). COX 2 and iNOS are both implicated in the pathophysiologic process of cartilage degeneration. For both, a statistical trend for repression of cytokine-induced synthesis was observed. The ability of GLN to regulate expression of these genes and their 153 respective inflammatory mediators has been demonstrated previously in equine cartilage (Fenton et al 2000a,b, Orth et a1 2002, Fenton et al 2002). We attribute the lack of a clearly demonstrable protective effect of GLN to the inherent variability in this design, as recent reports using bovine explants have shown that GLN at 5 ug/ml resulted in significant inhibition of rhIL-l B-induction of NO and PGE2 (Chan et al 2004). The most common form of AP-l is as a heterodimer of two proteins, c-Jun and c-Fos (Smeal et al 1989). IN K phosphorylates and hence activates c-Jun, which then translocates to the nucleus and dimerizes with c-fos (Firestein et al 1999). The expression of JNK increases in OA cartilage (Geng et al 1996), with elevated expression preceding the expression of degradative enzymes such as the MMPS along with clinical signs of OA (Han et al 1998). Due to inconsistent induction of the JNK transcript by reIL-10 in this study, the statistical analysis was limited to data from only two horses. Thus, despite what appears to be a dramatic repression of cytokine-induced expression of this protein with 10 ug/ml GLN, categorical conclusions cannot be drawn. The inconsistent induction of JNK by IL-1 in this study may be dose related, as maximal induction of JNK activity required 10 ng/ml rhIL-l in rabbit articular chondrocytes (Scherele et al 1997). In contrast to our findings, two previous studies utilizing much higher doses of GLN found no influence on AP-l DNA binding or activity, despite a concurrent suppression of NFKB (Largo et al 2003, Gouze et al 2002). Nonetheless, the possibility that GLN influences JNK synthesis warrants further investigation and could provide an additional mechanism by which GLN can inhibit IL-l mediated effects in OA cartilage. 154 While we were unable to demonstrate a significant effect of GLN on a marked induction of NFKB expression by reIL-18 (P = 0.11), it remains possible that at least some of the effects of GLN are effected via this intracellular signaling pathway. Using human osteoarthritic cartilage, Largo et al reported that glucosamine sulfate significantly inhibited IL-l induced NFKB DNA binding, translocation of p50 and p65 subunits of NFKB to the nucleus, as well as preventing IL-l mediated degradation of IKB, the natural inhibitor of NFKB (Largo et al 2003). However, doses used were at least 100 fold greater, and doses equivalent to those used in this study had no effect, suggesting that the influence of GLN on this transcription factor may be dose dependent. Regulation of gene expression was observed with GLN but not for CS. The effects of GLN on pre-translational regulation of genes coding for proteins implicated in OA has been the subject of a number of studies, however the potential effects of CS at the level of gene expression have been less frequently examined. Nonetheless, our results of finding no significant effect of CS on cytokine-stimulated equine chondrocytes contradict a number of previous reports utilizing higher doses of CS (Orth et al 2002, Bassleer et al 1998a). Specifically, using bovine cartilage explants, CS at 20 ug/ml significantly reduced the production of PGE2 and NO in 24-hour cultures (Chan et al 2004). The specific reasons for this difference remain to be elucidated but may reflect differences in species (bovine vs. equine), experimental design (pellets vs. explants), the form of CS employed (source, purity, molecular weight), the magnitude of the arthritogenic stimulus (50 ng/ml rhIL-lB vs 0.05 ng/ml reIL-113) and/or the influence of timing of supplementation (pre-incubation vs simultaneous to IL-l). Conversely, the beneficial 155 effects of CS may be principally related to anabolic processes, rather than preventing catabolic ones. For example, CS enhances the expression of genes such as type II collagen, suggesting that its chondroprotective effects may be in part due to stimulation of collagen and proteoglyearn/glycosaminoglycan synthesis (Bassleer et al 1998a, Nishikawa et al 1985, Conte et al 1991, Ronca et al 1998). Osteoarthritis research using in vitro models of cartilage degradation incorporating lipopolysaccharide, crude mixtures of inflammatory mediators contained in conditioned media, or recombinant cytokines to induce cartilage degradation represents a popular and cost-effective means of investigating pathophysiologic events and the effects of putative therapeutic agents on the disease process. Both glucosamine and chondroitin sulfate have been investigated in this manner; however, many of the studies to date have used relatively large quantities of both arthritogenic stimulus and nutraceuticals (Fenton et al 2000a,b, Orth et al 2002, Byron et al 2003, Bassleer et al 1998a, Nishikawa et al 1985, Largo et al 2003, Gouze et a1 2002, Fenton et al 2002). This series of experiments were planned to address these limitations of previous studies. The design employed both a sub- saturating dose of recombinant cytokine and physiologically relevant concentrations of the potential cartilage sparing preparations. Though arguably more biologically relevant than some previously conducted work, the experiments were complicated by considerable variability from several sources. We endeavored to utilize a dose of reIL-l B to produce a half-maximal response in our genes of interest. The dose of 500 pg/ml was determined by preliminary experiments employing 4 of our genes of interest using tissue from 5 different horses in an attempt to address the substantial intrinsic variability among horses. 156 Despite these efforts, the response to reIL-113 in the subsequent experiments varied widely at both the gene and animal level. As such, the pre-requisite 2-fold up-regulation of most genes was not unifome achieved, resulting in inferential analyses disadvantaged by relatively small numbers. The analysis of relative gene expression used herein was adapted from the 2(-AACT) method (Livak et al 2001). Validation of this technique is necessary for each experimental model as certain requirements need to be adhered to, including similarity in expression levels and amplification efficiencies of the target and reference (housekeeping) genes, along with stability of housekeeping gene expression. Variation in housekeeping gene stability has been documented in a number of studies, prompting recommendations to use more than one gene for normalization (V andesompele et al 2002, Suzuki et al 2000). As housekeeping genes have not been previously exhaustively compared using equine cartilage, and considerable variability was observed during initial amplifications using RNA isolated from GLN and CS treated pellets, we conducted a number of ancillary experiments to more fully characterize both amplification efficiencies and stability of a variety of standard reference genes including 185, B-actin, Bz-microglobulin, GAPDH, RPL19, and ubiquitin. We observed that the suitability of each housekeeping gene varied depending on the gene of interest and between horses. Similar sizeable inter-individual differences have been documented in studies conducted in other species (Dheda et al 2004, Tricarico et al 2002). A geometric mean (V andesompele et al 2002) of 18s, Bz-microglobulin and GAPDH was used in an attempt to incorporate requirements for expression levels, amplification efficiency and gene 157 stability for particular genes of interest and each individual horse. However, inconsistent and irregular amplification behavior may have hampered our attempts to detect subtle treatment effects using this model. Despite attempts to limit the effects of an observed lack of constitutive expression on the part of the housekeeping genes, there remained important dispersion in the data set. It would appear that for experiments of this type provisions need to be made for greater replication than has been typical of experiments using more potent stimulation and pharmacologic doses of therapeutic compounds. 158 Footnotes 8 Gibco, Grand Island, New York b Sigma, St. Louis, MO ° Roche, Indianapolis, Indiana d TRIzol®, Invitrogen, Carlsbad, CA 6 Rneasy mini kit, Qiagen, Valencia, CA fNanodrop, g Invitrogen, Carlsbad, CA h Perkin-Elmer Applied Biosystems, Foster City, CA iOperon Technologies, Alameda, CA; and Macromolecular Structure, Sequencing and Synthesis Facility, East Lansing, MI j Genbank Database, National Centre Biotechnology Information k Glucosamine Hydrochloride, Sigma, St. Louis, MO ' Chondroitin Sulfate A, Sigma, St. Louis, MO m SigmaStat version 2.0 159 H<< 34mm UEOGfififl§¢ How wow: macaw finance—850: mo A.mxl.mv 328568 .8th 8.628 can cation "Am unassuv m 033—. 160 50558 0508 5055555050055 50055550505050 9 320:8; :5 582m mumz 505.5 555550050059 50000555550558 $3.85 m ;22 A8558 0:38 8 50555050050500 505000050000 329:2 :5 83555 a .32 50550550050055 05505050505099 5%: m ;22 05500555500500 05050050055500 3 855 N ;22 0 50500500005005 55505505050500 $325 2 52 50553 58:: 9 5500005500500 55505000055005 3.22:2 55 $3335 0;: 55505055555550 5005050050000 $585 522 50500000550500 55000050555050 E 55 N 50 5005050500508 5005005505055 8&85 N 55 555000555000 555555005005 $385 _ wm< heath omho>om hoamhfi thaEch Iona-=— Ecmmm000< Juan—=00 0:09 .5582 550 0508309 083-38 0358:5255 5% com: 3.235 .5 macaw .5 A.mxl.mv 50558 5th 0532 EB 5535,.“ Am sauna—EV : 03:. 161 12 .. mo”_”_Pw___-___w_s__-_¢_J_hmm__ J b .____s________. _, CN‘O’Q II ll ll i? l N ll l l l l p___. “—j“ I L T T 0 10 50 100 500 1000 5000 10000 20000 Dose reIL-1B (pg/ml) Mean Relative Expression Figure 1 (chapter 3): Relative expression of COX 2 to graduated doses of recombinant interleukin-10 (reIL-10). Chondrocyte pellet cultures (3 x 106 cells/pellet) were exposed to graduated concentrations (0, 10, 50, 100, 500, 1000, 5000, 10000, and 20000 pg/ml) of reIL-10 for 6 hours followed by RNA isolation and quantitative PCR using specific primers for COX 2. An approximately half-maximal stimulation was evident at 500 pg/ml. A qualitatively similar response was observed for Agg 1, iNOS and MMP l3. 8 ytg V _—h__#-fi 36 ————— 7 — —~— 7 —— a. ase- — i we — — -—— ——a O41_ 7 i i - _fi __ *___#_L_.___._ 5 %3 F—« — — i*——_ _'_— fl (:2 T f 5‘“* *1 “-5 ”4*“ W'fl * ‘“ s ‘* “ r" 0 r T T I connol 500 1000 5000 10000 reIL-18(pglml) Figure 2 (chapter 3): Relative expression of COX 2 to graduated doses of recombinant interleukin-10 (reIL-10). Chondrocyte pellet cultures (6 x 106 cells/pellet) were exposed to graduated concentrations (0, 500, 1000, 5000, and 10000 pg/ml) of reIL- 10 for 12 hours followed by RNA isolation and quantitative PCR using specific primers for COX 2. A qualitatively similar response was observed for iNOS. 162 0.6 Log", Relative Expression 0.8 - 0.4 0.2 «7 onions __fi -L -___ ___s_:__ *r I .. Control IL-1 IL-1+2.Suglml lL-1 +5ug/ml GS lL-1+1009/ml GS GS Treatment Figure 3 (chapter 3): Influence of graduated concentrations of glucosamine hydrochloride on recombinant interleukin-IB-induced MMP 13 expression. Values are mean (+/- SEM) from experiments conducted with tissue from 3 different horses. Relative expressions were calculated using the 2(-AACT) method with 188, GAPDH and [32microglobulin as the reference genes. * Indicates statistical significance at p < 0.05 compared to positive control. L091. Relative Gene Expression 0.8 - 0.6 « 0.4 ~ Coiltrol lL-1 Treatment w __ fl _ __ . in- ,_ F‘_l .J. lL-1 + 2.5ug/ml lL-1 + Sug/ml GS IL-1 + 10uglml GS GS Figure 4 (chapter 3): Influence of graduated concentrations of glucosamine hydrochloride on recombinant interleukin-IB-induced Agg 1 expression. Values are mean (+/- SEM) from experiments conducted with tissue from 4 different horses. Relative expressions were calculated using the 2(-AACT) method with 185, GAPDH and 02microglobulin as the reference genes. * Indicates statistical significance at p < 0.05 compared to positive control. 163 1.4 - r: .9. g 1.2 .. — —»— ---._ ”—— ——~ “v“ WW— _____mm- 3 1 A— — ——«— fl -.#v:~____-_4. __mr_ ..... ___. 2 0.8 ~* W i w, fl .. —’ .#...._____ _~ _-____‘_-._ #w- __ 8 06~ 55555 in ___ _. -5 __ -__,_____ e 5 0.4 T.--_-_,_ ——— « — ———— % 02 E — — _ J —._ s .5 I “é o _, g _ _ __ l_.l_l §' OZI l - ' Control lL-1 lL-1 + 2.5ug/ml IL-1 + Suglml GS lL-1 + 1OUg/ml GS GS Treatment Figure 5 (chapter 3): Influence of graduated concentrations of glucosamine hydrochloride on recombinant interleukin-IB-induced iNOS expression. Values are mean (+/- SEM) from experiments conducted with tissue from 3 different horses. Relative expressions were calculated using the 2(-AACT) method with 188, GAPDH and [32microglobulin as the reference genes. There is a statistical trend for a treatment effect (P = 0.06). 1- 0.8« ——— ‘ 7. _.__________ - O4 4‘7 -~——— -—r—~ 0,2 «*7 _...__f____ ._ “a... L” ..... " "H o _ ‘_.I.W_ [_—_l l—-h—l Log“, Relative Gene Expression -02 _ Control lL-1 lL-1 + 2.Suglml lL-1 + Suglml lL-1 + 10uglm| GS GS GS Treatment Figure 6 (chapter 3): Influence of graduated concentrations of glucosamine hydrochloride on recombinant interleukin-1 B-induced COX 2 expression. Values are mean (+/- SEM) from experiments conducted with tissue from 3 different horses. Relative expressions were calculated using the 2(-AACT) method with 18s, GAPDH and [32microglobulin as the reference genes. There is a statistical trend for a treatment effect (P = 0.08). 164 References Adebowale A, Du J, Liang Z, et al. The bioavailability and pharmacokinetics of glucosamine hydrochloride and low molecular weight chondrotin sulfate after single and multiple doses to beagle dogs. Biopharm Drug Dispos 2002;23:217-25. Aigner T, Zien A, Gehritz A, et al. Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology. Arthritis Rheum 2001;44:2777-89. Bassleer CT, Combal JP, Bougaret S, et al. Effects of chondroitin sulfate and interleukin- 1 beta on human articular chondrocytes cultivated in clusters. Osteoarthritis Cartilage 1998;6:196-204. Bassleer C, Rovati L, Franchimont P. Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritis articular cartilage in vitro. Osteoarthritis Cartilage 1998;6:427-34. Byron CR, Orth MW, Venta PJ, Lloyd J W, Caron JP. Influence of glucosamine on matrix metalloproteinase expression and activity in lipopolysaccharide-stimulated equine chondrocytes. Am J Vet Res 2003;64:666-71. Caron JP, Tardif G, Martel-Pelletier J, et al. Modulation of matrix metalloprotease 13 (collagenase 3) gene expression in equine chondrocytes by IL-1 and corticosteroids. Am J Vet Res 1996;57:1631-4. Chan PS, Caron JP, Rosa GJM, et al. Pharmacologically relevant concentrations of glucosamine and chondroitin sulfate regulate gene expression and synthesis of nitric oxide and prostaglandin E2 in vitro. Osteoarthritis Cartilage (submitted 2004). Clegg PD, Carter SD. Matrix metalloproteinase-2 and —9 are activated in joint disease. Equine VetJ 1999;31:324-30. Conte A, de Bernardi M, Palmieri L, et al. Metabolic fate of exogenous chondroitin sulfate in man. Arzneim-Forsch Drug Res 1991;41:768-72. 165 Cook JL, Anderson C, Kreeger JM, et al. Effects of human recombinant interleukin-10 on canine articular chondrocytes in three-dimensional culture. Am J Vet Res 2000;7z766- 70. Dheda K, Huggett J, Bustin S, et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 2004;37: 112-9. Dodge GR, Jimenez SA. Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes. Osteoarthritis Cartilage 2003;11:424-32. Du J, Eddington N. Determination of the chondroitin sulfate disaccharides in dog and horse plasma by HPLC using chondroitinase digestion, precolumn derivatization, and fluorescence detection. Analytical Biochemistry 2002;306:252-8. Du J, White N, Eddington ND. The bioavailability and pharmacokinetics of glucosamine hydrochloride and chondroitin sulfate after oral and intravenous single dose administration in the horse. Biopharm Drug Dispos 2004; 25: 109-16. Fenton JI, Chlebek-Brown KA, Peters TL, et al. Glucosamine HCl reduced equine articular cartilage degradation in explant culture. Osteoarthritis Cartilage 2000;8:258-65. Fenton JI, Chlebek-Brown KA, Peters TL, et al. The effects of glucosamine derivatives on equine articular cartilage degradation in explant culture. Osteoarthritis Cartilage 2000;8:444-51. Fenton JI, Chlebek KA, Caron JP, et al. Effect of glucosamine on interleukin-1 conditioned articular cartilage. Equine Vet J 2002 (Supp 34):219-23. Firestein GS, Manning AM. Signal Transduction and Transcription Factors in Rheumatic Disease. Arthritis Rheum l999;4:609-21. Geng Y, Valbracht J, Lotz M. Selective activation of the Mitogen-activated Protein Kinase Subgroups c-Jun NH2 Terminal Kinase and p38 by IL-1 and TNF in Human Articular Chondrocytes. J Clin Invest 1996; 98:2425-30. 166 Gouze JN, Bordji K, Gulberti S, et al. Interleukin-10 down-regulates the expression of Glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: Influence of Glucosamine on Interleukin-l B-medicated effects in rat chondrocytes. Arthritis Rheum 2001 :44:351-60. Gouze JN, Bianchi A, Bécuwe P, et a1. Glucosamine modulates IL-l-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-KB pathway. FEBS Lett 2002:510;166-70. Han Z, Boyle DL, Manning AM, et al. AP-l and NF-kappaB regulation in rheumatoid arthritis and murine collagen-induced arthritis. Autoimmunity 1998:28zl97-208. Kevorkian L, Young DA, Darrah C. Expression Profiling of Metalloproteinases and their Inhibitors in Cartilage. Arthritis Rheum 2004;50:131-41. Koshy PJ T, Lundy CJ, Rowan AD, et al. The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin-1 and oncostatin M: A time course study using real time Quantitative Reverse Transcription- Polymerase Chain Reaction. Arthritis Rheum 2002;46:961-7. Largo R, Alvarez-Sofia MA, Diez—Ortego I, et al. Glucosamine inhibits IL-lB-induced NF KB activation in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 2003 :1 1;290—8. Liang Z, Leslie J, Adebowale A, et al. Determination of the nutraceutical, glucosamine hydrochloride, in raw materials, dosage forms and plasma using pre-column derivatization with ultraviolet HPLC. J Pharm Biomed Analysis 1999;20:807-814. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-8. Mengshol JA, Vincenti MP, Brinckerhoff CE. IL-1 induces collagenase-3 (MMP 13) promoter activity in stably transfected chondrocytic cells: requirement for Runx-Z and activation by p38 MAPK and JNK pathways. Nuc Acids Research 2001;29:4361-72. MacDonald MH, Stover SM, Willits NH, et al. Regulation of matrix metabolism in equine cartilage explant cultures by interleukin 1. Am J Vet Res 1992;53:2278-85. 167 Martel-Pelletier J, Alaaeddine N, Pelletier JP. Cytokines and their role in the pathophysiology of osteoarthritis. Front Biosci l999;4:D694-703. Mengshol JA, Vincenti MP, Coon CI, et al. Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-JUN N- terminal kinase and nuclear factor KB: Differential regulation of collagenase 1 and 3. Arthritis Rheum 2000;43:801-11. Morris EA, Treadwell BV. Effect of interleukin 1 on articular cartilage from young and aged horses and comparison with metabolism of osteoarthritic cartilage. Am J Vet Res 1994;55:138-46. Nagase H, Woessner F. Matrix metalloproteinases. Minireview. J Biol Chem 1999;274:21491-4. Nakamura H, Shibakawa A, Tanaka M et al. Effects of glucosamine hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteases by chondrocytes and synoviocytes in osteoarthritis. Clin Exp Rheumat 2004;22:293-9. Nishikawa H, Mori I, Umemoto J. Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane. Arch Biochem Biophys 1985;240:146-53. Orth MW, Peters TL, Hawkins JN. Inhibition of articular cartilage degradation by glucosamine-HCI and chondroitin sulphate. Equine Vet J 2002;3:224-9. Pavelka K, Gatterova J, Olejarova M, et al. Glucosamine sulfate use and delay of progression of knee OA: A 3-year, randomized, placebo-controlled, double blind study. Arch Intern Med 2002;162:21 13-23 Pelletier JP, DiBattista JA, Roughley P, et al. Cytokines and inflammation in cartilage degradation. Rheum Dis Clin North Am 1993;19:545-68. 168 Reginster JY, Deroisy R, Rovatti LC, et al. Long—term effects of glucosamine sulphate on osteoarthritis progression: a randomized, placebo-controlled clinical trial. Lancet 2001;357:251-6. Reno C, Marchuk L, Sciore P et al. Rapid isolation of total RNA from small samples of hypocellular, dense connective tissues. Biotechniques 1997;22:1082-6. Richardson D, Dodge GR. Effects of interleukin-10 and tumor necrosis factor-a on expression of matrix-related genes by cultured equine articular chondrocytes. Am J Vet Res 2000;61:624-30. Ronca F, Palmieri L, Panicucci P, et al. Anti-inflammatory activity of chondroitin sulfate. Osteoarthritis Cartilage 1998 (Suppl 6A);14-21. Rosselot G, Reginato AM, Leach RM. Development of a serum-free system to study the effect of growth hormone and insulinlike growth factor-I on cultured postembryonic growth plate chondrocytes. In Vitro Cell Dev Biol 1992;A:235-44. Salminen HJ, Saamanen AM, Vankemmelbeke MN, et al. Differential expression patterns of matrix metalloproteinases and their inhibitors during development of osteoarthritis in a transgenic mouse model. Ann Rheum Dis 2002;61:591-7. Sandy JD, Gamett D, Thompson V, et al. Chondrocyte mediated catabolism of aggrecan: aggrecanase dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine. Biochem J 1998;335:59-66. Sasaki H, Hattori T, Fujisawa T, et al. Nitric oxide mediates interleukin-1 induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultures rabbit articular chondrocytes. Endocrinology 1996;137:3729-37. Scherle PA, Pratta MA, Feeser WS, et al. The effects of IL-1 on mitogen-activated protein kinases in rabbit articular chondrocytes. Biochem Biophys Res Commun 1997;230:573-7. Smeal T, Angel P, Meek J, et al. Different requirements for formation of Juanun and Jun:Fos complexes. Genes Dev 1989;3z2091-100. 169 Suzuki T, Higgins P, Crawford DR. Control selection for RNA quantitation. Biotechniques 2000;29:332-7. Thompson CC, Clegg PD, Carter SD. Differential regulation of gelatinases by transforming growth factor beta-1 in normal equine chondrocytes. Osteoarthritis Cartilage 2001;9:325-31. Tricarico C, Pinzani P, Bianchi S, et al. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 2002;309:293-300. Tung JT, Fenton J1, Arnold C, et al. Recombinant equine interleukin-10 induces putative mediators of articular cartilage degradation in equine chondrocytes. Can J Vet Res 2002;66:19-25. Vandesompele J, De Preter K, Pattyn F, et al. Accurate noramlization of real-time quantitative RT—PCR data by geometric averaging of multiple internal control genes. Genome Biology 2002;3:0034.1-OO34.1 l Verbruggen G, Goemaere S, Veys EM. Chondroitin sulfate: S/DMOAD (structure/disease modifying anti-osteoarthritis drug) in the treatment of finger joint OA. Osteoarthritis Cartilage 1998;6 (Suppl A):37-8. Vincenti MP, Brinckerhoff CE. Early response genes induced in chondrocytes stimulated with the inflammatory cytokine interleukin-10. Arthritis Res 2001;3:381-8. Vincenti MP, Brinckerhoff CE. Transcriptional regulation of collagenase (MMP 1, MMP 13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis Res 2002;4:157-64. 170 CHAPTER 4: QUANTIFICATION OF GENE EXPRESSION USING A SUBSATURATING MODEL OF OSTEOARTHRITIS: CONSIDERATIONS FOR ENDOGENOUS CONTROLS AND EXPERIMENTAL DESIGN Quantitative real time polymerase chain reaction (Q-RT-PCR) is a powerful tool for determining gene expression. This technique is inherently more sensitive than reverse transcriptase polymerase chain reaction (PCR) and Northern hybridization, as gene expression is quantified as the reaction proceeds rather than at the end point. An additional advantage is the ability to measure gene expression in smaller samples (Fehr et al 2000). Problems with end point analysis reside fiom the influence that factors other than the initial amount of template may have on the end point of the reaction. Such factors include consumption of nucleotides, product degradation and depletion of reagents as the reaction nears completion, which can compromise precision, sensitivity and resolution. Furthermore, the end point of a PCR reaction varies between samples, and quantification with gel electrophoresis may not enable resolution of such variability in yield. In contrast, Q-RT-PCR uses fluorescence detection methods to measure the amount of PCR product at each cycle, creating an amplification plot of accumulated product over the reaction. This enables quantification during the exponential phase when reagents are not depleted and reaction kinetics favor doubling of the PCR product with every cycle. However, inherent requirements of Q-RT-PCR need to be adhered to. Endogenous control genes, or housekeeping genes, are used for normalization of quantity of input RNA, loading error, variation in RNA integrity, and efficiency of cDNA conversion and 171 PCR amplification. Validity of the delta delta Ct method (Livak et a1 2001), commonly used for quantification of gene expression by Q-RT-PCR, relies on certain assumptions. The expression of the endogenous control gene should remain constant, and the amplification efficiencies of the target gene and the endogenous control must be approximately equal. Notably, the control gene should maintain a consistent level of expression and not be altered by the experimental treatment. Recently, a number of reports have documented problems with a number of frequently used endogenous control genes (Suzuki et al 2000, Thellin et al 1999). This may reflect their documented functions in vivo, but also may be attributed to the effect of experimental conditions or pathological processes on the expression of these genes. The expression of housekeeping genes may vary among species, tissues and cell types related to differences in overall transcriptional activity. For instance, glyceraldehyde phosphate dehydrogenase (GAPDH) is an important glycolytic enzyme, catalyzing the oxidative phosphorylation of glyceraldehydes-3-phosphate, with other roles in endocytosis, DNA replication and repair, and apoptosis documented (Sirover 1997). However, expression may be altered by a variety of in vitro and in vivo conditions such as hypoxia, insulin and dexamethasone (Oliveira et al 1999). Cellular proliferation is an important modulator of GAPDH levels, with cell-cycle dependent regulation apparent in quiescent cell cultures that are stimulated by addition of serum (Suzuki et al 2000). B-actin, often used as a housekeeping gene, is a ubiquitous cytoskeletal protein; however, expression levels can vary following hypoxia, and exposure of cultured cells to growth 172 factors or serum (Elder et a1 1984). Further, the presence of pseudogenes may interfere with mRNA quantification (Dimhofer et al 1995). Ribosomal RNAs, such as 185 and 28s, constitute 85-90% of toal cellular RNA and are often used as endogenous controls. However, ribosomal RNA may not always be representative of mRNA levels as rRNA may be lost during purification of mRNA, and rRNA is not recommended for quantification of samples when oligo dT sequences are used for cDNA synthesis (Overbergh et al 2003). Also, expression levels of rRNA are much higher compared to mRNA (Bustin 2002). This series of experiments were designed to investigate the effect of glucosamine and chondroitin sulfate on expression of a number of genes of interest in equine pellet cultures stimulated with a sub-saturating dose of recombinant equine interleukin-113 (reIL-10). However, results were hampered by both variability in the response to reIL-10 at both the gene and animal level, and variabililty in housekeeping gene expression despite equalization of input template and validation procedures. In an attempt to address limitations of previous studies that had used large quantities of both interleukin and/or nutraceuticals (Fenton et al 2000a,b, Orth et al 2002, Byron et al 2003, Bassleer et al 1998, Nishikawa et a1 1985, Largo et al 2003, Gouze et al 2002, Fenton et a1 2002), experiments employed both a sub-saturating dose of recombinant cytokine and physiologically relevant concentrations of glucosamine and chondroitin sulfate. Other potential confounding variables in experimental design were also addressed in an attempt to more accurately mimic in vivo conditions. For instance, we used serum-free media as the potential influence of undefined serum factors on experimental parameters has been 173 identified in a number of studies (Barnes et al 1980, Rosselot et al 1992, Kawcak et al 1996). Further, the influence of serum on housekeeping gene expression has been quantified (Elder et al 1984, Schmittgen et al 2000, Suzuki et al 2000). Similarly, pellet cultures were chosen based on established phenotypic stability and a more accurate reflection of chondrocytes in vivo compared to other culturing techniques (Stewart et al 2000) Preincubation with GS and CS prior to stimulation with reIL-10 was chosen in an attempt to identify whether these commonly used compounds could potentially prevent induction of inflammatory mediatiors and degradative enzymes and hence OA. Pretreatment with glucosamine has been beneficial in other in vitro studies using rat and human chondrocytes, albeit using higher doses (Largo et al 2003, Gouze et al 2002). Whilst the duration of establishment of pellet cultures prior to experimental manipulation did change during the course of the study, this was not thought to have influenced our results, as housekeeping gene expression varied in cultures of different ages. Although a number of experiments were conducted in S-day old cultures, the extracellular matrix of pellet cultures is established by day 3 with aggrecan and collagen content approximating in vivo levels by day 4 (Stewart et al 2000). Initially, RNA was isolated following a 6-hour incubation period, based on previous findings of temporal aspects of induction by reIL-10 in our laboratory (Tung et a1 2002). However, considerable variability both at the gene and animal level in terms of both response to reIL-10 stimulation and response to treatment with glucosamine and 174 chondroitin sulfate was apparent. Normalization to GAPDH, 183 and the geometric average of both GAPDH and 18s produced a quantitatively similar result, as did normalization to the median housekeeping CT and analysis of results using IL-l as the calibrator. Erroneus outlier results were common, and in conjunction with the wide variability that was apparent, further hindered analysis of results with no statistically significant effect of treatment discemable. This prompted the change in experimental design to a 12-hour incubation. Despite these efforts, the variation in terms of response to reIL-113 and to treatment with glucosamine and chondroitin sulfate in the subsequent experiments remained. As such, the pre-requisite 2-fold up-regulation of most genes was not uniformly achieved, and the variability between individuals was marked. Although limited experiments were conducted using glucosamine and chondroitin sulfate in combination, results were again hampered by considerable variability, resulting in insufficient samples for analysis. Initially, experiments were performed following a 6- hour incubation with doses of GS and CS of 100 ;1le alone or in combination. These doses were chosen to approximate those that had been previously shown to be effective using bovine cartilage in our laboratory. Although data was only collected from 2 horses, in general both GLN and CS appeared to negatively influence expression of MMP l3, COX 2 and Agg 1, results that were not consistent with responses to these nutraceuticals demonstrated previously. This experiment was performed before the impact of variability in housekeeping gene expression was investigated. However, the combination of GLN and CS did appear to reduce NF KB expression and CS alone may have had a stimulatory effect on type II collagen production, although accuracy of the latter effect was hindered 175 by a comparable expression of positive and negative controls. Conversely, the combination at doses of GLN 10 ug/ml and CS 20 ;1ng following a lZ-hour incubation had no effect. The dose of 20 ug/ml was chosen based on doses that had been shown to be effective in combination in bovine cartilage (Orth et al 2004). As previously discussed, the lack of effect of CS may relate to a number of factors inherent to the design of the experiments. In particular, the CS used in bovine cartilage experiments in our laboratory previously was obtained from a different source, and it has recently been suggested that purified low molecular weight CS may be more effective than intact CS (Cho et al 2004). The analysis of relative gene expression used herein was adapted from the 2(-AACT) method (Livak et al 2001). Validation of this technique is necessary for each experimental model as certain requirements need to be adhered to, including similarity in expression levels and amplification efficiencies of the target and reference (housekeeping) genes, along with stability of housekeeping gene expression. Variation in housekeeping gene stability has been documented in a number of studies, prompting recommendations to use more than one gene for normalization (Hamalainen et a1 2001, Vandesompele et al 2002, Suzuki et a1 2000, Schmid et al 2003). In particular, the use of a ribosomal gene in conjunction with another housekeeping gene has been advocated (Ropenga et al 2004). Other methods proposed include normalization to total RNA content; however, this does not account for differences in reverse transcriptase or PCR amplification efficiencies (Bunner et a1 2004). To minimize variations in reverse transcriptase efficiency, all samples from a particular horse were reverse transcribed 176 simultaneously. In an attempt to correct for variability, Q-RT-PCR was conducted based equalization of both RNA and cDNA concentration quantified with spectrophotometry. However, variability in both housekeeping gene expression and genes of interest remained, even when cDNA content was further quantified with Nanodrop® technology to further ensure accurate sample loading. Levels of gene expression for available housekeeping genes varied substantially (Figure 1) and there were substantial differences in individual housekeeping gene stability between horses (Figure 2). These results were also apparent with the 6-hr incubation period, and, although only 185 and GAPDH were investigated at that time period, these genes showed greater variability than at the 12-hour period. Amplification efficiency also varied for each housekeeping gene (Table II). The effect of normalization with different housekeeping genes alone or in combination was determined for MMP 13 using tissue from 5 horses, including using the best housekeeping gene for each horse based on least variability. The effect on MMP 13 expression was marked, highlighting the potential for confounding that could be introduced if an inappropriate housekeeping gene was chosen. As the ideal choice of housekeeping gene varied not only for each gene of interest but also between horses, three housekeeping genes (1 8s, GAPDH and Bz-microglobulin) were subsequently chosen in an attempt to satisfy all criteria based on expression level, amplification efficiency, and the lowest level of variability, both overall and for each horse. Using the geometric average of 4 housekeeping genes did not appear to be any more beneficial than the geometric average of 3 housekeeping genes. 177 Six housekeeping genes (185, B-actin, Bzmicroglobulin, GAPDH, RPL19, ubiquitin) were selected based on the availability of full or partial length equine sequences for commonly used endogenous controls. No equine nucleotide sequences were available for other commonly used housekeeping genes such as 285, acidic ribosomal protein, [5- glucuronidase, cyclophilin A, hypoxanthine guanine phosphorylbosyl transferase (HPRT), hypoxanthine ribosyl transferase, phosphoglycerokinase or transferrin receptor. The three housekeeping genes subsequently used in this series of experiments have been used for normalization in Q-RT-PCR, PCR and Northern hybridizarion experiments using equine cartilage conducted in our and other laboratories. However, these genes have not been previously exhaustively compared. Normalization should reduce variability associated with errors in RNA purity, RNA quantification, or the amount of RNA initially used (Balaburski et al 2003). We observed that the suitability of each housekeeping gene varied depending on the gene of interest and between horses, suggesting that the housekeeping genes varied independently of each other and necessitating the use of more than one gene as an endogenous control. Similar sizeable inter-individual differences have been documented in studies conducted in other species (Dheda et al 2004, Tricarico et al 2002). A geometric mean (V andesompele et al 2002) of 18s, Bz-microglobulin and GAPDH was used in an attempt to incorporate requirements for expression levels, amplification efficiency and gene stability for particular genes of interest and each individual horse. However, inconsistent and irregular amplification behavior may have hampered our attempts to detect subtle treatment effects using this model, as there needs to be less than one AC’T difference in order to detect differences in gene expression lower than two fold (Livak et al 2001, Vandesompele et a1 2002). 178 Despite attempts to limit the effects of an observed lack of constitutive expression on the part of the housekeeping genes, there remained important dispersion in the data set. Interestingly, such large gene variability has been identified in other studies investigating expression of degradative enzymes and extracellular matrix proteins in OA cartilage (Aigner et al 2001, Martin et al 2001). Areas requiring further investigation include the effects of using the sub-saturating dose of IL-1 simulataneously with physiologically relevant doses of GLN and CS; using different forms of CS; and the duration of effect of GLN and CS. Further, the effect of GLN and CS on expression of other genes for which primers were also developed (aggrecan, type II collagen, ILRa, TIMP 1, 3), remains to be determined, as does the effect on other transcription factors and interrnediarys in IL-1 signaling pathways and pathophysiological phenomena such as apoptosis. 179 h<< Sara A8558 030E << mOZw PU805“? =80qu OUEUOHEUOOEUE0m 00.5.5 cheetah 1.38:2 :e_mm000< 0.55.50 0:00 .5502 £20 00205.23 085.30. 03:53:05 8.“ 00m: 00:0m t5 A.mxl.mv 805308 BEE; 0232 0:0 Eazcom "G. noun—anev u 030,—. 180 Table 11 (chapter 4): Suitability of housekeeping genes for each gene of interest based on amplification efficiency. The value of the slope of the regression of the plot of ACT versus log cDNA dilution was used as a measure of the difference in amplification efficiencies between the gene of interest and each housekeeping gene. A difference in efficiency of