Histologic evidence for a humoral immune response in synovitis associated with cranial cruciate ligament disease in dogs.

OBJECTIVE: To investigate histopathological features of synovium from dogs with cranial cruciate ligament disease (CCLD) to seek mechanisms of osteoarthritis (OA) associated with CCLD. STUDY DESIGN: Retrospective, single-institution case series. ANIMALS: Thirty client-owned dogs. METHODS: Synovial biopsies (n = 30) obtained from stifles with CCLD were assessed by using two synovitis histopathology grading systems (Krenn and Hospital for Special Surgery [HSS]). The Krenn synovitis score was interpreted as "no synovitis," "low-grade," or "high-grade," while inflammatory subtype (low, mixed, or high) was determined by a computational algorithm within the HSS system. Comparison of synovitis scores was based on degree of CCL rupture and presence of meniscal tears. RESULTS: Histopathological changes and synovitis scores were similar regardless of degree of rupture (partial n = 5, complete n = 25) or presence of meniscal injury (n = 12) and were characterized by hyperplastic and lymphoplasmacytic synovitis with increased vascularity (30/30) and the presence of hemosiderin deposits (28/30), binucleated plasma cells (28/30), mucoid change (25/30), and Mott cells (16/30). Thirteen (43%) specimens were consistent with high-grade synovitis according to the Krenn system, while 11 (37%) specimens fit into the high-inflammatory subtype with the HSS system. CONCLUSION: Synovitis associated with canine CCLD in this study population was lymphoplasmacytic and was often highly inflammatory, with the presence of cells pertaining to humoral immunity. Humoral immune responses may play key roles in the synovitis associated with CCLD. CLINICAL SIGNIFICANCE: Modulation of biological factors that provoke humoral immune responses may mitigate symptoms of OA that persist and progress even after surgical treatment of CCLD in dogs.

A dietary polysaccharide from Eucheuma cottonii downregulates proinflammatory cytokines and ameliorates osteoarthritis-associated cartilage degradation in obese rats.

Osteoarthritis (OA) is a common form of arthritis, which is characterized by the degeneration of articular cartilage, leading to joint dysfunction. Oral drug therapy seems to ameliorate some signs and symptoms of OA, but may be accompanied by side effects and does not appear to be effective long-term. Seaweed has received much attention for pharmacological application due to its various biomedical properties, including the anti-inflammation, antitumor, and antioxidant effects. This study investigated the ameliorative effects of a dietary polysaccharide from Eucheuma cottonii extract (ECE) on an anterior cruciate ligament transection with partial medial meniscectomy surgery (ACLT+MMx) to induce OA in high-fat diet (HFD)-induced obese rats. Male Sprague-Dawley rats were fed an HFD for 12 weeks before ACLT+MMx surgery, after which they were administered a daily oral gavage of saline (Sham, OB Sham, and OBOA) and either low-dose ECE (100 mg per kg body weight), high-dose ECE (400 mg per kg body weight), or glucosamine sulfate as a positive control (OBOAGS; 200 mg per kg body weight) for 5 weeks. Treatment with ECE decreased the body weight, triglyceride and total cholesterol (TC) levels, and the TC/high-density lipoprotein (HDL)-C ratio in the obese rats. Additionally, ECE downregulated the expression of proinflammatory cytokines, including tumor necrosis factor-α, interleukin-1ß, and leptin, and suppressed nuclear factor-kappa B and extracellular-signal-regulated kinase-1/2 expression, resulting in a decrease in the levels of matrix metalloproteinase (MMP)-1 and MMP-13 and prostaglandin-E2 and attenuated cartilage degradation. These results demonstrate that the dietary polysaccharide from ECE can suppress OA development in obese rats, suggesting its potential efficacy as a promising candidate for OA treatment.

Inflammatory pattern of the infrapatellar fat pad in dogs with canine cruciate ligament disease.

BACKGROUND: Despite the importance of inflammation during the pathogenesis of cranial cruciate ligament disease (CCLD) in dogs and despite the latest knowledge suggesting a significant role of adipose tissue in osteoarthritis, the infrapatellar fat pad (IFP) was up to now mostly disregarded in veterinary investigations. In the present study, the inflammatory activity of the IFP, the main adipose structure within the stifle joint, was thoroughly investigated to evaluate its potential impact in the pathogenesis of this common disease of our canine companions. Samples of IFP, subcutaneous adipose tissue (ScAT) of the thigh and synovial fluid in both diseased (n = 36) and healthy control (n = 23) dogs were tested for their immune cell composition but also for interleukins (IL-1ß, IL-6, IL-8, IL-10), degradative enzymes (MMP-1, MMP-3, MMP-13, TIMP-2, iNOS) and adipokines (leptin and adiponectin). Characterization of the immune cell composition was ascertained by fluorescence activated cell sorting. Gene expression and protein release of the inflammatory markers was determined by real RT-qPCR and ELISA. RESULTS: IFPs of dogs with CCLD had a significantly increased immune cell count with T cells (CD3) as the most abundant immune cells. T cells and macrophages (CD14) were significantly increased compared to healthy controls or corresponding ScAT. In addition, IFPs of dogs with CCLD demonstrated a significant increase on gene as well as protein level of multiple inflammatory indicators (IL-1ß, IL-6, MMP-1, MMP-13) compared to the other tissues. TNFα was only increased on gene expression. Adipokine analysis showed higher secretion of adiponectin and lower leptin secretion in IFP from dogs with CCLD than from controls. In the synovial fluid from dogs with CCLD concentrations of IL-1ß, MMP-1, MMP-13 as well as leptin were significantly increased compared to the synovial fluid from healthy control dogs. CONCLUSIONS: The present study indicates that the IFP is a potential contributory factor in the pathogenesis of CCLD, due to its inflammatory phenotype and the proximity within the stifle joint. To determine the extent of this possible inter-relationship, further studies need to be undertaken.

Cruciate ligament degeneration and stifle joint synovitis in 56 dogs with intact cranial cruciate ligaments: Correlation of histological findings and numbers and phenotypes of inflammatory cells with age, body weight and breed.

The majority of dogs with ruptured cranial cruciate ligaments (crCLs) have inflammatory changes of the stifle joint suggesting that synovitis is an important factor which is involved in the development of degenerative ligament changes. Detailed information is not available concerning the possible occurrence of inflammatory changes in the stifle joint synovium of dogs with macroscopically intact crCLs and its correlation with presence and severity of degenerative changes of the crCLs. Therefore, the purpose of this study was to examine post mortem tissue samples of 56 dogs with non-ruptured crCLs histologically for the presence of inflammatory and degenerative changes of the stifle joint synovium and cruciate ligaments, respectively. In 25/56 (44.6%) dogs, histology showed that both lymphoplasmacytic synovitis and degenerative alterations of the crCLs were present. In these dogs, there was a significant positive statistical correlation between the severity of synovitis and degenerative crCL lesions. The results suggest that synovitis in the stifle joints of dogs with non-ruptured crCLs is a frequent event and probably is involved in the development of degenerative lesions occurring in canine crCLs. Also, the severity of crCL degeneration in these 25 dogs was significantly correlated with their age and body weight. In 2/56 (3.6%) cases, only lymphoplasmacytic synovitis was found in the absence of degenerative crCL lesions. In 15/56 (26.8%) dogs, only degenerative lesions of the crCLs without synovitis were present. Statistically, a significant correlation was found between the severity of degenerative alterations and age and body weight of these dogs. Phenotyping of inflammatory cells by immunohistochemistry showed that the synovium of dogs which histologically had lymphoplasmacytic synovitis was infiltrated with CD3+ T lymphocytes, CD79+ B lymphocytes, major histocompatibility class II antigen (MHC class II)+ cells and macrophages expressing CD163 or S100/A8/S100A9 (calprotectin), while tartrate-resistant acid phosphatase (TRAP)+ cells were absent. Quantification and statistical evaluation of inflammatory cell types in the inflamed synovium revealed that the numbers of lymphocytic cells and macrophages were significantly correlated with the severity of synovitis. These findings indicate that, besides T and B lymphocytes, both pro- and anti-inflammatory macrophages play a role in the development of degenerative crCL alterations.

Radiographic and magnetic resonance imaging predicts severity of cruciate ligament fiber damage and synovitis in dogs with cranial cruciate ligament rupture.

Cruciate ligament rupture (CR) and associated osteoarthritis (OA) is a common condition in dogs. Dogs frequently develop a second contralateral CR. This study tested the hypothesis that the degree of stifle synovitis and cranial cruciate ligament (CrCL) matrix damage in dogs with CR is correlated with non-invasive diagnostic tests, including magnetic resonance (MR) imaging. We conducted a prospective cohort study of 29 client-owned dogs with an unstable stifle due to complete CR and stable contralateral stifle with partial CR. We evaluated correlation of stifle synovitis and CrCL fiber damage with diagnostic tests including bilateral stifle radiographs, 3.0 Tesla MR imaging, and bilateral stifle arthroscopy. Histologic grading and immunohistochemical staining for CD3+ T lymphocytes, TRAP+ activated macrophages and Factor VIII+ blood vessels in bilateral stifle synovial biopsies were also performed. Serum and synovial fluid concentrations of C-reactive protein (CRP) and carboxy-terminal telopeptide of type I collagen (ICTP), and synovial total nucleated cell count were determined. Synovitis was increased in complete CR stifles relative to partial CR stifles (P<0.0001), although total nucleated cell count in synovial fluid was increased in partial CR stifles (P<0.01). In partial CR stifles, we found that 3D Fast Spin Echo Cube CrCL signal intensity was correlated with histologic synovitis (SR = 0.50, P<0.01) and that radiographic OA was correlated with CrCL fiber damage assessed arthroscopically (SR = 0.61, P<0.001). Taken together, results of this study show that clinical diagnostic tests predict severity of stifle synovitis and cruciate ligament matrix damage in stable partial CR stifles. These data support use of client-owned dogs with unilateral complete CR and contralateral partial CR as a clinical trial model for investigation of disease-modifying therapy for partial CR.

Histological and immunohistological analysis of degenerative changes in the cranial cruciate ligament in a canine model of excessive tibial plateau angle.

OBJECTIVE: To create a canine model of excessive tibial plateau angle (eTPA) and assess the chondroid metaplasia and extracellular matrix alteration in the cranial cruciate ligament. METHODS: Seven mature female Beagles were included. Cylindrical osteotomy was performed bilaterally in the proximal tibia. The TPA was increased to approximately 40° in the left tibia (eTPA stifle) and left unchanged in the right tibia (control stifle). Exercise stress was started at three months postoperatively, and at 12 months postoperatively the dogs were euthanatized and the cranial cruciate ligaments were collected. The specimens were subjected to haematoxylin and eosin staining to assess the ligamentocyte morphology and immunostaining to assess the type I (COLI), type II (COLII), and type III (COLIII) collagen, and the sry-type HMG box 9 (SOX9) staining. RESULTS: Macroscopic cranial cruciate ligament injury was absent in six dogs but present in the eTPA stifle of one dog, which was excluded from the analysis. The ligamentocyte density decreased and the percentage of round ligamentocytes increased in the eTPA stifles. The COLII, COLIII, and SOX9 staining increased significantly and COLI deposition decreased in the eTPA stifles compared to the control stifle. CLINICAL SIGNIFICANCE: The extracellular matrix changed, COLI deposition decreased, and COLIII and SOX9 staining increased in the cranial cruciate ligament of the eTPA stifles. SOX9 may contribute to COLII synthesis in the extracellular matrix of the cranial cruciate ligament in eTPA stifles, and eTPA may promote chondroid metaplasia and extracellular matrix alteration.

Anti-inflammatory response of dietary vitamin E and its effects on pain and joint structures during early stages of surgically induced osteoarthritis in dogs.

There is evidence that vitamin E (VE) has anti-inflammatory and analgesic properties in human osteoarthritis (OA). This double-blinded and randomized pilot study used a broad spectrum of clinical and laboratory parameters to investigate whether such beneficial effects could be detected in a canine experimental OA model. Dogs were divided into 2 groups: control (n = 8), which received a placebo, and test group (n = 7), which received 400 IU/animal per day of VE for 55 d, starting the day after transection of the cranial cruciate ligament. Lameness and pain were assessed using a visual analogue scale (VAS), numerical rating scale (NRS), and electrodermal activity (EDA) at day 0, day 28, and day 55. Cartilage and synovial inflammation lesions were assessed. One-side comparison was conducted at an alpha-threshold of 10%. At day 56, dogs were euthanized and concentrations of prostaglandin E2 (PGE2), nitrogen oxides (NOx), and interleukin-1 beta (IL-1ß) were measured in synovial fluid. Concentrations of NOx and PGE2 in synovial fluid were lower in the test group (P < 0.0001 and P = 0.03, respectively). Values of VAS, NRS, and EDA showed a consistent trend to be lower in the test group than in the control, while statistical significance was reached for VAS at day 55 and for EDA at day 28 (adjusted P = 0.07 in both cases). Histological analyses of cartilage showed a significant reduction in the scores of lesions in the test group. This is the first time that a study in dogs with OA using a supplement with a high dose of vitamin E showed a reduction in inflammation joint markers and histological expression, as well as a trend to improving signs of pain.

La vitamine E (VE) est connue par ses propriétés anti-inflammatoires et analgésiques dans le traitement de l'ostéoarthrose (OA) chez l'humain. Dans notre étude pilote nous avons utilisé un ensemble de paramètres cliniques et de laboratoire afin de déterminer si ces effets bénéfiques de la VE pourront être détectés chez le chien arthrosique, dans un modèle expérimental d'OA. Les chiens utilisés ont été divisés en 2 groupes: témoin (n = 8), qui a reçu un placebo et un groupe supplémenté (n = 7), qui a reçu 400 UI de VE/animal/jour pendant 55 jours, la supplémentation orale a commencé un jour après la section du ligament croisé crânial. Avant la chirurgie (J0), J28 et J55 après chirurgie, la boiterie et la douleur ont été évaluées à l'aide d'une échelle visuelle analogique (EVA), d'une échelle d'évaluation numérique (NRS), et par la mesure de l'activité électrodermique (EDA). Les lésions au niveau du cartilage et l'inflammation synoviale ont été évalués. Une seule comparaison statistique a été réalisée avec un seuil alpha à 10 %. Au jour 56, les chiens ont été euthanasiés et les concentrations de prostaglandine E2 (PGE2), d'oxyde d'azote (NOx) et d'interleukine-1 bêta (IL-1ß) ont été mesurées dans le liquide synovial. Les concentrations synoviales de NOx et de PGE2 étaient plus faibles dans le groupe traité (P < 0,0001 et P = 0,03, respectivement). Les valeurs de l'EVA, de NRS et de l'EDA ont montré une tendance constante à être plus faible dans le groupe traité par comparaison au groupe témoin, avec un effet significatif de la VE qui a été observé pour VAS au jour 55 et EDA au jour 28 (P ajustée = 0,07 dans les deux cas). Les analyses histologiques du cartilage ont montré une réduction significative des scores lésionnels chez le groupe traité. Cette étude est la première à démontrer qu'une supplémentation orale avec une dose élevée de VE chez des chiens arthrosiques permet de réduire la libération des marqueurs inflammatoires et les lésions histologiques au niveau du cartilage, ainsi qu'une tendance à améliorer les signes de douleur.(Traduit par Docteur Serge Messier).

Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis.

OBJECTIVE: In experimental collagenase-induced osteoarthritis (OA) in the mouse, synovial lining macrophages are crucial in mediating joint destruction. It was recently shown that adipose-derived stem cells (ASCs) express immunosuppressive characteristics. This study was undertaken to explore the effect of intraarticular injection of ASCs on synovial lining thickness and its relation to joint pathology in experimental mouse OA. METHODS: ASCs were isolated from fat surrounding the inguinal lymph nodes and cultured for 2 weeks. Experimental OA was induced by injection of collagenase into the knee joints of C57BL/6 mice. OA phenotypes were measured within 8 weeks after induction. Histologic analysis was performed, and synovial thickening, enthesophyte formation, and cartilage destruction were measured in the knee joint. RESULTS: ASCs were injected into the knee joints of mice 7 days after the induction of collagenase-induced OA. On day 1, green fluorescent protein-labeled ASCs were attached to the lining layer in close contact with macrophages. Thickening of the synovial lining, formation of enthesophytes associated with medial collateral ligaments, and formation of enthesophytes associated with cruciate ligaments were significantly inhibited on day 42 after ASC treatment, by 31%, 89%, and 44%, respectively. Destruction of cartilage was inhibited on day 14 (65%) and day 42 (35%). In contrast to early treatment, injection of ASCs on day 14 after OA induction showed no significant effect on synovial activation or joint pathology on day 42. CONCLUSION: These findings indicate that a single injection of ASCs into the knee joints of mice with early-stage collagenase-induced OA inhibits synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction.

Update on the aetiopathogenesis of canine cranial cruciate ligament disease.

Cranial cruciate ligament disease (CCLD) is the most common cause of hindlimb lameness in the dog, being associated with and eventually leading to stifle osteoarthritis. Canine cranial cruciate ligament disease is a gradual degeneration of the ligament extracellular matrix (ECM) leading to ligament rupture. The aetiopathogenesis of this condition is still poorly understood but several risk factors have been identified such as breed, bodyweight, gender and conformation. Recent developments in this area include the role of genetics, stifle joint conformation, ligament ECM metabolism, and inflammation associated with immune-mediated disease within the stifle joint. A genetic mode of inheritance has been demonstrated in the Newfoundland which is predisposed to CCLD. Increased cellular metabolism within the cranial cruciate ligament has been directly associated with increased craniocaudal stifle joint laxity in dog breeds at high risk of CCLD. Conformation abnormalities, such as a narrowed distal femoral intercondylar notch, in high risk breeds have been shown to be associated with alterations in cranial cruciate ligament ultrastructure. Increased production of inflammatory cytokines, such as cathepsins and interleukins, by the stifle synovial cells may occur secondary to or may be an inciting cause of ligament degeneration. Future research endeavours will focus on the association between immune-mediated response and fibrocartilaginous metaplasia and matrix degradation within the cranial cruciate ligament, and whether this can be altered in all susceptible dogs or only certain breeds.

Modulation of inflammation by vitamin E and C supplementation prior to anterior cruciate ligament surgery.

Muscle atrophy commonly follows anterior cruciate ligament (ACL) injury and surgery. Proinflammatory cytokines can induce and exacerbate oxidative stress, potentiating muscle atrophy. The purpose of this study was to evaluate the influence of prior antioxidant (AO) supplementation on circulating cytokines following ACL surgery. A randomized, double-blind, placebo-controlled trial was conducted in men undergoing ACL surgery, who were randomly assigned to either: (1) AO (200 IU of vitamin E (50% d-alpha-tocopheryl acetate and 50% d-alpha-tocopherol) and 500 mg ascorbic acid), or (2) matching placebos (PL). Subjects took supplements twice daily for 2 weeks prior to and up to 12 weeks after surgery. Each subject provided five blood samples: (1) baseline (Bsl, prior to supplementation and approximately 2 weeks prior to surgery), (2) presurgery (Pre), (3) 90 min, (4) 72 h, and (5) 7 days postsurgery. Following surgery, inflammation and muscle damage increased in both groups, as assessed by increased circulating IL-6, C-reactive protein, and creatine kinase. During AO supplementation, plasma alpha-T and AA increased while gamma-T concentrations decreased significantly (P< 0.05). At 90 min the AO group displayed a significant decrease in AA, an inverse correlation between AA and (interleukin) IL-8 (r(2)= 0.50, P< 0.05), and a significantly lower IL-10 response than that of the PL group. IL-10 was significantly elevated at 90 min and 72 h in the PL group. In summary, our findings show that circulating inflammatory cytokines increase and AO supplementation attenuated the increase in IL-10 in patients post-ACL surgery.

Contributions of different intraarticular tissues to the acute phase elevation of synovial fluid MMP-2 following rat ACL rupture.

Inflammation and accumulation of matrix metalloproteinases (MMPs) in synovial fluids may be involved in the poor healing ability of the anterior cruciate ligament (ACL) after injury. With a rat ACL rotating injury model, we found that levels of IL-1beta, IL-6, and TNF-alpha were significantly higher in synovial fluids after ACL injury. MMP-2 activity and global MMP activity in synovial fluids also increased significantly in a time-dependent manner. Ex vivo studies showed that all tissues contributed to the elevation of MMP-2 in synovial fluids, especially synovium and the injured ACL. We concluded that although the regular wound-healing mechanism also occurs after ACL injury, accumulation of MMP activity in the synovial fluids, due to all of the intraarticular tissues, may be at least one of the important reasons why an injured ACL cannot be repaired.

Immunopathological mechanisms in dogs with rupture of the cranial cruciate ligament.

The majority of studies on cranial cruciate ligament (CrCL) disease to date have been carried out on dogs that already sustained a CrCL rupture, which is the end-stage of the disease. Investigations have recently been carried out to study humoral and cellular immunopathological mechanisms in predisposed dogs before clinical rupture of the contralateral CrCL. The cruciate ligaments are mainly composed of collagen type I, and immune responses to collagen have been suggested as a cause of CrCL degradation in dogs. None of these investigations showed evidence that anticollagen type I antibodies alone initiate CrCL damage. However, in predisposed dogs a distinct anticollagen type I antibody gradient was found towards the contralateral stifle joint that eventually sustained a CrCL rupture, suggesting that there was an inflammatory process present in these joints before detectable joint instability occurred. The importance of cellular reactivity to collagen type I in cruciate disease also remains unclear. Peripheral blood mononuclear cell proliferation to collagen type I was very diverse in dogs with cruciate disease whereas some sham operated dogs and healthy dogs tested positive as well. It is not yet determined whether cellular reactivity to collagen type I exists locally in the stifle joints nor whether this could initiate CrCL degradation. Inflammatory processes within the stifle joint can alter the composition of the cruciate ligaments. In animal models of immune-mediated synovitis, the mechanical strength of the CrCL is significantly reduced. Immunohistochemical studies on synovial tissues from dogs with rheumatoid arthritis and dogs with cruciate disease revealed that the pathologic features are similar in both joint pathologies and that the differences are mainly quantitative. Joint inflammation induced by biochemical factors such as cytokines has been implied in CrCL degeneration. In several studies, the levels of pro-inflammatory and T helper cytokines were measured in dogs that sustained a CrCL rupture, but the exact role of the various cytokines in the pathogenesis of CrCL disease remains inconclusive. More recently, the levels of the cytokines have been investigated over time in predisposed dogs before and after CrCL rupture. IL-8 expression tended to be higher in stifle joints that will rupture their CrCL during the next 6 months than in those that will not, indicating an inflammatory process in these joints before clinical rupture. This review provides a comprehensive overview of all possible implications of humoral and cell-mediated immune responses published in dogs with cruciate disease together with publications from human joint diseases. Furthermore, this review highlights recent findings on cytokines and proteinases in the accompanying joint inflammation.

Ligament repair: a molecular and immunohistological characterization.

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during "ligamentization". Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction.

Cytokine mRNA expression in synovial fluid of affected and contralateral stifle joints and the left shoulder joint in dogs with unilateral disease of the stifle joint.

OBJECTIVE: To examine mRNA expression of cytokines in synovial fluid (SF) cells from dogs with cranial cruciate ligament (CrCL) rupture and medial patellar luxation (MPL) and determine mRNA expression for 3 joints (affected stifle, unaffected contralateral stifle, and left shoulder joints) in dogs with unilateral CrCL rupture. SAMPLE POPULATION: 29 stifle joints with CrCL rupture (29 dogs), 8 stifle joints with MPL (7 dogs), and 24 normal stifle joints (16 clinically normal dogs). PROCEDURES: Immediately before reconstructive surgery, SF was aspirated from the cruciate-deficient stifle joint or stifle joint with MPL. Fourteen of 29 dogs had unilateral CrCL rupture; SF was also aspirated from the unaffected contralateral stifle joint and left shoulder joint. Those 14 dogs were examined 6 and 12 months after reconstructive surgery. Total RNA was extracted from SF cells and reverse transcription-PCR assay was performed to obtain cDNA. Canine-specific cytokine mRNA expression was determined by use of a real-time PCR assay. RESULTS: Interleukin (IL)-8 and -10 and interferon-gamma expression differed significantly between dogs with arthropathies and dogs with normal stifle joints. For the 14 dogs with unilateral CrCL rupture, a significant difference was found for IL-8 expression. Before reconstructive surgery, IL-8 expression differed significantly between the affected stifle joint and left shoulder joint or contralateral stifle joint. Six months after surgery, IL-8 expression was significantly increased in the unaffected contralateral stifle joint, compared with the shoulder joint. CONCLUSIONS AND CLINICAL RELEVANCE: No conclusions can be made regarding the role of the examined cytokines in initiation of CrCL disease.

Lymphocyte proliferation to collagen type I in dogs.

The objective of this study was to investigate if cellular reactivity to collagen type I exists in dogs with unilateral cranial cruciate ligament (CrCL) rupture and if it relates to disease progression. The patient group consisted of 10 dogs with unilateral CrCL rupture. The control dogs consisted of three healthy control dogs, and two healthy dogs with unilateral sham operations of the stifle joint. All dogs were assayed repeatedly every 6 months for 12-24 months. Peripheral blood mononuclear cells were isolated from whole blood and were cultured with human collagen type I at concentrations of 5, 20 and 40 microg/ml for 6 and 7 days. Lymphocyte reactivity to collagen type I occurred not only in dogs with CrCL rupture, but also in sham-operated dogs and healthy dogs. Five of the eight assays (63%) performed at the time of operation or at the time of diagnosis of CrCL rupture had a stimulation index (SI) >or=3.0. This was not significantly different compared to healthy control dogs, not to the sham-operated control dogs. The CrCL rupture was assessed intraoperatively in six cases. Three cases had partial rupture and three had complete rupture. Only one dog with partial rupture, and two dogs with complete rupture had a positive SI. An increase in proliferation to collagen type I was seen in dogs with CrCL rupture, whereas it either remained stable or decreased in the control dogs. No distinct pattern in lymphocyte reactivity to collagen type I could be established from the dogs that sustained a CrCL rupture in the contralateral stifle joint, although most dogs that did not sustain a CrCL rupture in the contralateral stifle joint remained negative during this study with exception of one dog. Further research is required to determine whether cellular reactivity to collagen type I may play an initiating role in cruciate degradation.

Evaluation of anticollagen type I antibody titers in synovial fluid of both stifle joints and the left shoulder joint of dogs with unilateral cranial cruciate disease.

OBJECTIVE: To evaluate anticollagen type I antibodies in synovial fluid of the affected stifle joint, the contralateral stifle joint, and the left shoulder joint of dogs with unilateral cranial cruciate ligament (CrCL) rupture during an extended period of 12 to 18 months. ANIMALS: 13 client-owned dogs with CrCL rupture and 2 sham-operated dogs. PROCEDURES: All dogs were examined and arthrocentesis of all 3 joints was performed every 6 months after surgery. Synovial fluid samples were tested for anticollagen type I antibodies by use of an ELISA. RESULTS: Dogs with partial CrCL rupture had higher antibody titers than dogs with complete rupture. Six of 13 dogs ruptured the contralateral CrCL during the study, whereby higher antibody titers were found for the stifle joints than for the shoulder joint. Seronegative dogs or dogs with extremely low antibody titers and 2 dogs with high antibody titers did not sustain a CrCL rupture in the contralateral stifle joint. CONCLUSIONS AND CLINICAL RELEVANCE: In most dogs that had a CrCL rupture of the contralateral stifle joint, a distinct antibody titer gradient toward the stifle joints was detected, suggesting that there was a local inflammatory process in these joints. However, only a small number of sham-operated dogs were used to calculate the cutoff values used to determine the anticollagen type I antibody titers in these patients. Synovial fluid antibodies against collagen type I alone do not initiate CrCL rupture because not all dogs with high antibody titers sustained a CrCL rupture in the contralateral stifle joint.

Replacement of human anterior cruciate ligaments with pig ligaments: a model for anti-non-gal antibody response in long-term xenotransplantation.

BACKGROUND: Understanding anti-non-gal antibody response is of significance for success in xenotransplantation. Long-term anti-non-gal response in humans was studied in patients transplanted with porcine patellar tendon (PT) lacking alpha-gal epitopes, for replacing ruptured anterior cruciate ligament (ACL). METHODS: Porcine PTs were treated with recombinant alpha-galactosidase to eliminate alpha-gal epitopes and with glutaraldehyde for moderate cross-linking of collagen fibers. The processed pig PTs were implanted to replace ruptured ACL in patients. RESULTS: In five of six evaluable subjects, the xenografts have continued to function for over two years and passed all functional stability assessments. Thus, processed porcine PT seems to be appropriate for replacing ruptured human ACL. Enzyme-linked immunosorbent assay and Western blot studies indicated that all subjects produced anti-non-gal antibodies against multiple pig xenoproteins, but not against human ligament proteins. Production of anti-non-gal antibodies peaked two to six months posttransplantation and disappeared after two years. CONCLUSIONS: These antibodies contribute to a low-level inflammatory process that aids in gradual xenograft replacement by infiltrating host fibroblasts that align with the pig collagen "scaffold" and secrete collagen matrix. The assays monitoring anti-non-gal antibodies will help to determine whether long-term survival of live organ xenografts requires complete suppression of this antibody response.