A new tool for early diagnosis of rheumatoid arthritis using combined biomarkers; synovial MAGE-1 mRNA and serum anti-CCP and RF.

INTRODUCTION: rheumatoid arthritis (RA) is a common autoimmune disease with unknown etiology and pathogenesis. Biomarkers have the potential to aid in the clinical diagnosis of the disease, or to provide means of detecting early signs of the disease. Evaluating Melanoma associated antigen genes (MAGE-1) mRNA expression rate in synovial fluid cells and serum levels of anti-cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) for RA early diagnosis. METHODS: a total of 213 subjects were enrolled in the study, 135 RA patients and 78 normal subjects with traumatic knee joints (control group). Serum RF and anti-CCP were estimated quantitatively using ELISA. MAGE-1 mRNA expression rate was analyzed by RT-PCR. RESULTS: a significant increase in serum levels of RF IgM and anti-CCP in RA patients compared to the controls. A positively significant correlation was found between serum anti-CCP and RF IgM. The expression rate of MAGE-1 mRNA was 100% in RA patients versus the controls (0%). The specificity and the sensitivity of the three biomarkers was 100%. CONCLUSION: the high expression rate of MAGE-1 in synovial fluid cells of RA patients is encouraging its utilization as a diagnostic biomarker for RA. The combined use of MAGE-1 transcript in synovial fluid cells, serum RF and anti-CCP is recommended for improving early diagnostic ability of RA.

Short-Term Outcomes of Percutaneous Trephination with a Platelet Rich Plasma Intrameniscal Injection for the Repair of Degenerative Meniscal Lesions. A Prospective, Randomized, Double-Blind, Parallel-Group, Placebo-Controlled Study.

Meniscal tears are the most common orthopaedic injuries, with chronic lesions comprising up to 56% of cases. In these situations, no benefit with surgical treatment is observed. Thus, the purpose of this study was to investigate the effectiveness and safety of percutaneous intrameniscal platelet rich plasma (PRP) application to complement repair of a chronic meniscal lesion. This single centre, prospective, randomized, double-blind, placebo-controlled study included 72 patients. All subjects underwent meniscal trephination with or without concomitant PRP injection. Meniscal non-union observed in magnetic resonance arthrography or arthroscopy were considered as failures. Patient related outcome measures (PROMs) were assessed. The failure rate was significantly higher in the control group than in the PRP augmented group (70% vs. 48%, P = 0.04). Kaplan-Meyer analysis for arthroscopy-free survival showed significant reduction in the number of performed arthroscopies in the PRP augmented group. A notably higher percentage of patients treated with PRP achieved minimal clinically significant difference in visual analogue scale (VAS) and Knee injury and Osteoarthritis Outcome Score (KOOS) symptom scores. Our trial indicates that percutaneous meniscal trephination augmented with PRP results in a significant improvement in the rate of chronic meniscal tear healing and this procedure decreases the necessity for arthroscopy in the future (8% vs. 28%, P = 0.032).

Serum Mast Cell Tryptase as a Marker of Posttraumatic Joint Contracture in a Rabbit Model.

OBJECTIVES: Mast cells have been identified as key mediators of posttraumatic joint contracture, and stabilizing medications (ketotifen) have been shown to decrease contracture severity. Serum mast cell tryptase (SMCT) levels are used clinically to monitor mast cell-mediated conditions. The goals of this study were to determine if SMCT levels are elevated in the setting of joint contracture, if they can be decreased in association with ketotifen therapy, and if they correlate with contracture severity. METHODS: This study used a previously developed rabbit model in which 39 animals were divided into 4 groups: operatively created joint contracture (ORC, n = 13), operatively created contracture treated with ketotifen at 2 doses (KF0.5, n = 9; KF1.0, n = 9), and healthy rabbits (NC, n = 8). Range of motion measures were performed at 8 weeks after the surgery. Serum samples were collected on postoperative days 1, 3, 5, 7, 21, 35, and 49. SMCT levels were measured using a rabbit-specific enzyme-linked immunosorbent assay. RESULTS: Levels of SMCT were highest in the operatively created joint contracture group and were significantly greater compared with both ketotifen groups (P < 0.001). Levels were highest at postoperative day 1 with a trend to decrease over time. A positive correlation between SMCT levels and contracture severity was observed in all operative groups (P < 0.05). CONCLUSIONS: Levels of SMCT are elevated in the setting of joint contracture, decreased in association with ketotifen therapy, and positively correlated with contracture severity. This is the first study to establish a relationship between SMCT and joint injury. Measurement of SMCT may be valuable in identifying those at risk of posttraumatic joint contracture.

Matrix metalloproteinase activity and prostaglandin E2 are elevated in the synovial fluid of meniscus tear patients.

PURPOSE: Meniscus tears are a common knee injury and are associated with the development of post-traumatic osteoarthritis (OA). The purpose of this study is to evaluate potential OA mediators in the synovial fluid and serum of meniscus tear subjects compared to those in the synovial fluid of radiographic non-OA control knees. MATERIALS AND METHODS: Sixteen subjects with an isolated unilateral meniscus injury and six subjects who served as reference controls (knee Kellgren-Lawrence grade 0-1) were recruited. Twenty-one biomarkers were measured in serum from meniscus tear subjects and in synovial fluid from both groups. Meniscus tear subjects were further stratified by tear type to assess differences in biomarker levels. RESULTS: Synovial fluid total matrix metalloproteinase (MMP) activity and prostaglandin E2 (PGE2) were increased 25-fold and 290-fold, respectively, in meniscus tear subjects as compared to reference controls (p < 0.05). Synovial fluid MMP activity and PGE2 concentrations were positively correlated in meniscus tear subjects (R = 0.83, p < 0.0001). In meniscus tear subjects, synovial fluid levels of MMP activity, MMP-2, MMP-3, sGAG, COMP, IL-6, and PGE2 were higher than serum levels (p < 0.05). Subjects with complex meniscus tears had higher synovial fluid MMP-10 (p < 0.05) and reduced serum TNFα and IL-8 (p < 0.05) compared to other tear types. CONCLUSIONS: Given the degradative and pro-inflammatory roles of MMP activity and PGE2, these molecules may alter the biochemical environment of the joint. Our findings suggest that modulation of PGE2 signaling, MMP activity, or both following a meniscus injury may be targets to promote meniscus repair and prevent OA development.

The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis.

BACKGROUND: The complement system is suggested to be involved in the pathogenesis of osteoarthritis (OA), and proinflammatory cytokines may play a role in OA development by inducing proteases. The association between complement factors, cytokines and OA has not been investigated. The aim of the present study was to explore the involvement of the complement system after knee trauma and in OA. METHODS: C4d, C3bBbP and soluble terminal complement complex (sTCC) resulting from complement activation were immunoassayed in synovial fluid from subjects with healthy knees (reference), OA, rheumatoid arthritis (RA; positive control), pyrophosphate arthritis (PPA; positive control) and knee injury; other biomarkers were previously assessed. Magnetic resonance imaging was used to assess joint injuries. RESULTS: Compared with levels in the reference group, the median concentrations of C4d, C3bBbP and sTCC in the OA, RA, PPA and knee injury groups were 2- to 34-fold increased (p < 0.001 to p = 0.044). For the knee injury group, the median concentrations of C4d, C3bBbP and sTCC were 5- to 12-fold increased (p < 0.001) at the day of injury; after 3-12 weeks, C3bBbP and sTCC concentrations were similar to reference levels; and C4d was still increased several years after injury. In the 0-12 weeks period after injury, the concentrations of C4d, C3bBbP and sTCC correlated positively with levels of interleukin (IL)-1ß, IL-6 and tumour necrosis factor α (r s range 0.232-0.547); none of the measured complement factors correlated with proteolytic fragments of aggrecan or cartilage oligomeric matrix protein. Knees with osteochondral fracture, with or without disrupted cortical bone, had higher concentrations of C4d (p = 0.014, p = 0.004) and sTCC (p = 0.004, p < 0.001) compared with knees without fractures. CONCLUSIONS: The complement system is activated in OA and after knee injury. Following knee injury, this activation is instant and associated with inflammation as well as with the presence of osteochondral fractures.

Circulating interleukin-6 is not altered while γ-tocopherol is increased in subjects scheduled for knee surgery with low vitamin D.

The purpose of this study was to identify if circulating interleukin (IL)-6 and γ-tocopherol (γT) fluctuate with vitamin D status in subjects with an underlying knee joint injury or disease. We hypothesized that low vitamin D associates with an increase in plasma γT while serum IL-6 remains unchanged in subjects with an underlying knee joint trauma or disease. Fifty-four subjects scheduled to undergo primary, unilateral anterior cruciate ligament reconstructive surgery (ACL; n=27) or total knee arthroplasty (TKA; n=27) were studied. Circulating γT, α-tocopherol (αT), lipids (cholesterol and triglycerides), IL-6, and 25-hydroxyvitamin D (25(OH)D) were measured in fasting blood samples obtained prior to surgery. Subjects were classified as vitamin D deficient, insufficient, or sufficient if they had a serum 25(OH)D concentration <50, 50-75, or >75nM, respectively. The majority (57%) of the subjects possessed a serum 25(OH)D less than 50nM. Circulating cholesterol, triglycerides, and IL-6 were not significantly (all p>0.05) different between vitamin D status groups. However, lipid corrected αT was significantly (p<0.05) decreased and both lipid- and non-lipid-corrected plasma γT concentrations were significantly (both p<0.05) increased with low serum 25(OH)D (i.e., <50nM). A significant (p<0.05) multi-variate analysis revealed that an increase in plasma γT per lipids was significantly (p<0.05) predicted by a decrease in serum 25(OH)D but not by a decrease in plasma αT per lipids. We conclude that low vitamin D associates with an increase in plasma γT but not IL-6 in subjects with an underlying joint injury or disease.

The incidence of deep venous thrombosis before arthroscopy among patients suffering from high-energy knee trauma.

PURPOSE: The purpose of the present study was to analyse the incidence of deep venous thrombosis (DVT) before knee arthroscopy in patients who had sustained high-energy knee injuries. METHOD: This study included 64 patients who underwent arthroscopic knee surgery as a result of injury from a traffic accident or a high fall. Venography was performed on the injured leg of each patient before arthroscopy. The patients were divided into two groups based on whether they had DVT. Correlation analysis was performed to determine the factors associated with DVT. RESULTS: A total of 32 (50 %) of the 64 patients had venographic evidence of DVT. Of these DVTs, seven were proximal (10.9 %). The D-dimer (DD) level was significantly higher in the DVT group, especially among the patients whose symptoms had persisted for more than 10 days. DVT is difficult to diagnose solely based on clinical symptoms, as some patients are symptomatic while others exhibit symptoms that could be attributed to trauma. CONCLUSIONS: The incidence of DVT before knee arthroscopy in patients with high-energy knee injuries was 50 %, and the prevalence of proximal DVT was 10.9 %. DD is a sensitive marker for DVT. No patient developed DVT with a DD level lower than 0.8 mg/L, but those with DD level higher than 1.5 mg/L had a much higher incidence of DVT developing in patients who had been injured for more than 10 days. A routine examination to exclude DVT in these patients should be performed before arthroscopy. LEVEL OF EVIDENCE: IV.

Acute Molecular Changes in Synovial Fluid Following Human Knee Injury: Association With Early Clinical Outcomes.

OBJECTIVE: To investigate whether molecules found to be up-regulated within hours of surgical joint destabilization in the mouse are also elevated in the analogous human setting of acute knee injury, how this molecular response varies between individuals, and whether it is related to patient-reported outcomes in the 3 months after injury. METHODS: Seven candidate molecules were analyzed in blood and synovial fluid (SF) from 150 participants with recent structural knee injury at baseline (<8 weeks from injury) and in blood at 14 days and 3 months following baseline. Knee Injury and Osteoarthritis Outcome Score 4 (KOOS4 ) was obtained at baseline and 3 months. Patient and control samples were compared using Meso Scale Discovery platform assays or enzyme-linked immunosorbent assay. RESULTS: Six of the 7 molecules were significantly elevated in human SF immediately after injury: interleukin-6 (IL-6), monocyte chemotactic protein 1, matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinases 1 (TIMP-1), activin A, and tumor necrosis factor-stimulated gene 6 (TSG-6). There was low-to-moderate correlation with blood measurements. Three of the 6 molecules were significantly associated with baseline KOOS4 (those with higher SF IL-6, TIMP-1, or TSG-6 had lower KOOS4 ). These 3 molecules, MMP-3, and activin A were all significantly associated with greater improvement in KOOS4 over 3 months, after adjustment for other relevant factors. Of these, IL-6 alone significantly accounted for the molecular contribution to baseline KOOS4 and change in KOOS4 over 3 months. CONCLUSION: Our findings validate relevant human biomarkers of tissue injury identified in a mouse model. Analysis of SF rather than blood more accurately reflects this response. The response is associated with patient-reported outcomes over this early period, with SF IL-6 acting as a single representative marker. Longitudinal outcomes will determine if these molecules are biomarkers of subsequent disease risk.

Evolution of C-reactive protein values in the first month after anterior cruciate ligament reconstruction: reference values.

PURPOSE: C-reactive protein (CRP) is often used as an infection marker in orthopaedic patients and in particular after anterior cruciate ligament (ACL) reconstruction surgery. The aim of this study is to obtain the reference values of CRP during the first month after an ACL reconstruction and to analyse the epidemiological and surgical parameters that affect these values. METHODS: One hundred and twenty ACL reconstructions were included. A CRP determination was performed preoperatively and 1, 7, 14, 21 and 28 days after surgery. CRP values under 5 mg/l were considered to be normal. RESULTS: One patient developed a septic arthritis in the second week postoperatively and was excluded. One hundred and seventeen patients [93 males and 24 females; mean age (standard deviation) 31.6 years (7.6)] underwent 119 ACL reconstructions with different techniques and grafts. Preoperative CRP (n = 119) was 1.80 mg/ml (2.6). Mean values at 1, 7, 14, 21 and 28 days were, respectively, 8.5 mg/ml (11.6), 10.5 mg/ml (17.0), 4.5 mg/ml (3.43), 4.4 mg/ml (7.59) and 3.4 mg/ml (3.03). Multivariate analysis showed that males had postoperative CRP levels 1.7 higher than females (p < 0.0001; 95 % CI 1.8-2.5); the patients operated by less experienced surgeons had levels 2.5 times higher than those operated by a highly experienced surgeons (p = 0.007; 95 % CI 1.2-3.4) and that if microfracture of a chondral lesion was associated, the levels increased 1.9 times (p = 0.021; 95 % CI 1.1-3.4). CONCLUSIONS: There are significant variations in CRP levels after ACL reconstruction in half of patients without infectious complications. Males, patients operated by less experienced surgeons and those with chondral lesions treated with microfracture had increased postoperative CRP levels. CRP values up to five times the normal limit are common in the month after an ACL reconstruction and are not necessarily associated with infection, especially in these groups.

Serum non-coding RNAs as biomarkers for osteoarthritis progression after ACL injury.

OBJECTIVE: The aim of this study was to examine serum non-coding RNAs as potential biomarkers for cartilage damage associated with anterior cruciate ligament (ACL) injury. METHODS: Serum was obtained from 80 patients 1 year after surgery for ACL injury and 60 normal donors without overt skeletal injury. Total serum RNA was isolated, small non-coding RNAs profiled by TaqMan array MicroRNA (miRNA) analysis and individual small RNA assays performed by quantitative TaqMan RT-PCR (qPCR). Semi-quantitative magnetic resonance imaging (MRI) analysis was performed using Whole Organ Magnetic Resonance Knee Score (WORMS) scoring for analysis of cartilage damage. RESULTS: Initial TaqMan array miRNA profiling showed an increased serum concentration of a small nucleolar RNA (snoRNA), U48, in five patients with cartilage damage compared with that in five patients without cartilage damage and six normal donors. Independent qPCR analysis of snoRNAs in serum from all patients and normal donors showed a strong association between the serum level of another snoRNA, U38, and cartilage damage in ACL injury patients and together with snoRNA, U48, clear distinction between ACL injury patients and normal donors. CONCLUSION: SnoRNAs U38 and U48 are significantly elevated in the serum of patients developing cartilage damage at 1 year after ACL injury. Serum levels of U38 have the potential to facilitate early diagnosis of patients with cartilage damage after ACL injury. This study suggests serum non-coding RNAs may serve as novel noninvasive biomarkers for the detection and assessment of cartilage damage after ACL injury.

Effects of rehabilitation treatment on thyroid function.

OBJECTIVE: The aim of the study was to evaluate the effects of an intensive rehabilitation programme on thyroid metabolism, the relationship between disability and thyroid hormone level, and the occurrence of nonthyroidal illness syndrome (NTIS) before and after rehabilitation. DESIGN, SUBJECTS AND MEASUREMENTS: This was a clinical prospective study. Orthopaedic surgery patients (n = 82) were classified into two groups: patients in whom early active mobilization and walking were possible (walking group, WG, n = 45), and patients in whom these were not recommended (nonwalking group, NWG, n = 37). Levels of free T3 (fT3), fT4, TSH and rT3 were measured before and after surgery, and then at 1, 3, 7, 14 and 30 days from the beginning of rehabilitation. Personal, nutritional and clinical data were acquired for all patients. The Barthel Index (BI) was used to assess disability before and after rehabilitation. RESULTS: Immediately after surgery, both groups of patients showed a significant decrease in mean fT3 concentrations and a significant increase in rT3; mean fT4 values decreased significantly only in NWG patients. Once rehabilitation had been completed, fT3 and rT3 levels returned to baseline values in WG patients. In NWG patients mean fT3 and fT4 levels continued to decrease significantly and rT3 values remained significantly high until the end of rehabilitation. NTIS occurred in 38% of the NWG patients. No significant changes in TSH levels were observed in either group. Finally, we observed a direct correlation between fT3 levels and the BI in WG patients. CONCLUSIONS: Our data suggest that early patient mobilization and physical activity during an active and intensive rehabilitation programme induce recovery of thyroid function and avoid occurrence of NTIS.

Increased serum levels of non-collagenous matrix proteins (cartilage oligomeric matrix protein and melanoma inhibitory activity) in marathon runners.

OBJECTIVE: Marathon runners have an increased risk of developing joint disease. During and after a 42-km run, elevation of multiple cytokines occurs in the blood, reflecting inflammatory processes. We compared this cytokine response with serum levels of cartilage oligomeric matrix protein (COMP) and melanoma inhibitory activity (MIA), two markers for joint metabolism and/or damage. METHODS: Serum from eight endurance-trained runners was collected shortly before the start of a marathon run, after 31 km, 42 km, 2 h after the end, on the first and on the second morning after the run. For comparison, serum was obtained from 35 healthy controls and 80 patients with knee joint injury, rheumatoid arthritis or osteoarthritis. Serum levels of C-reactive protein (CRP), interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1RA), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), soluble interleukin-6 receptor (sIL-6R, gp80), soluble tumor necrosis factor receptor II (sTNFRII, p75), COMP and MIA were measured by ELISA. RESULTS: Compared with healthy controls, the runner's baseline serum levels of TNF-alpha, sIL-6R, COMP and MIA were significantly increased. COMP and MIA levels, higher than the upper normal limits of 5 microg/ml and 6 ng/ml respectively, were found in seven and five of eight runners. The elevated levels of COMP were similar to those found in joint injury or osteoarthritis, and the elevated levels of MIA were comparable to those reported in rheumatoid arthritis. During the run, the serum levels of IL-1RA, IL-6, TNF-alpha and COMP rose significantly, and gradually returned to baseline within 24 h. Only modest changes of CRP, sIL-6R, sTNFRII and MIA occurred during the run. Late elevations of CRP and MIA were observed after 24 and 48 h. The correlation analysis suggests associations between COMP, sIL-6R, TNF-alpha, IL-1RA on one hand and sTNFRII, and MIA and CRP on the other hand. CONCLUSIONS: Elevated baseline levels of COMP and MIA might reflect increased joint matrix turnover and/or damage due to prior extreme physical training. During the run, COMP was increasing possibly due to the severe physical strain on joint structures, associated with the early inflammation. After the run, MIA and CRP increased within 24 h, suggesting a correlation with later inflammatory processes. Thus, our data suggest that COMP and MIA are markers for distinct aspects of joint metabolism and/or damage in both disease and sport.

Persistent high serum levels of cartilage oligomeric matrix protein in a subgroup of patients with traumatic knee injury.

The objective was to assess whether changes of cartilage oligomeric matrix protein (COMP) serum levels can predict the development of osteoarthritis following traumatic knee injury. Sera and synovial fluids were acquired at surgery (T0) and postoperatively during the first (T1) and second (T2) year from 30 knee-injured patients. COMP levels and anti-COMP autoantibodies were quantified by ELISA. Radiographs and patient questionnaires were used to assess outcomes. At T0, compared with controls (1.6 +/- 1.6 micrograms/ml), the serum COMP concentration was significantly elevated (6.5 +/- 2.8 micrograms/ml) with a tendency to further increase (T0 vs. T1, P = 0.076) and subsequently decrease (T1 vs. T2, P = 0.074). However, individual variations are observed, e.g. persistently high (8/30) or increasing (T0 to T2, 8/30) serum COMP. Ten of these patients have elevated COMP at T2 that increased from T0. COMP levels in serum and synovial fluid correlated significantly (P = 0.012). Interestingly, some patients who revealed increasing serum levels of COMP from T0 to T2 displayed anti-COMP autoantibodies. These data suggest that local immune response could contribute to further joint damage. The subgroup of 10 patients (33%) with elevated and increasing serum COMP levels and in particular the patients with antibodies against cartilage matrix molecules appear at increased risk for developing posttraumatic osteoarthritis.

[C-reactive protein as a postoperative parameter of infection in hip joint replacement and knee joint operations]. / CRP als postoperativer Infektionsparameter bei Hüftgelenksersatz- und Kniegelenksoperationen (Teil 1).

Prospectively 70 male patients with elective arthroplasty of the hip (n = 34) and with operation of the anterior cruciate ligament (n = 36) were studied in respect of the postoperative screening of C-reactive protein (CRP), the erythrocyte sedimentation rate (ESR), the white blood cell count and the clinical signs of infection. There were 9 infectious postoperative complications, all with an increase of CRP. But also 6 other patients with no clear infection showed a rise or a plateau of CRP, which could only be explained by the presence of a hematoma, an effusion or a subclinical infection. The close postoperative screening of CRP appeared to be quite costly and laborious, especially in a population with a low infection rate. We therefore suggest a CRP-screening only on patients or operative procedures with wellknown high risk. In addition we studied the determination of two CRP-levels with a short interval following the clinical suspect of a postoperative infection (described in part II of this paper).

Intraarticular ferritin-bound iron in rheumatoid arthritis. A factor that increases oxygen free radical-induced tissue destruction.

Iron mobilized from ferritin is able to convert superoxide and hydrogen peroxide, which are produced in large amounts in rheumatoid arthritis (RA), to the extremely toxic hydroxyl radical. We have found that synovial fluid ferritin is increased significantly in RA patients compared with levels in controls. The high synovial fluid:serum ferritin ratio is compatible with the hypothesis that synovial fluid ferritin is derived from the synovial membrane. We found no difference in ferritin concentrations in the synovial membranes of RA patients compared with those of controls. Quantitative data on the amount of iron bound to ferritin showed that the level was 2.9 times higher in RA synovial membranes than in those of controls. Moreover, RA synovial fluid contained considerable amounts of iron bound to ferritin. Calculation of the iron saturation of ferritin revealed that RA synovial membranes contained a mean of 2,210 moles of iron per mole of ferritin: a significant elevation when compared with the mean value of 1,500 moles found in the synovial membranes of the controls. The decreased saturation of ferritin in RA synovial fluid, compared with that in the synovial membrane, could be caused by an uncompensated release of iron from ferritin, which has been induced by superoxide that is produced by stimulated granulocytes. The results demonstrate that in the joints of RA patients, sufficient ferritin loaded with iron is available to stimulate oxygen free radical damage.