The Effects of Different Management Strategies or Rehabilitation Approaches on Knee Joint Structural and Molecular Biomarkers Following Traumatic Knee Injury: A Systematic Review of Randomized Controlled Trials for the OPTIKNEE Consensus.

OBJECTIVE: To summarize the effectiveness of management strategies and rehabilitation approaches for knee joint structural and molecular biomarker outcomes following anterior cruciate ligament (ACL) and/or meniscal tear. DESIGN: Intervention systematic review. LITERATURE SEARCH: We searched the MEDLINE, Embase, CINAHL, CENTRAL, and SPORTDiscus databases from their inception up to November 3, 2021. STUDY SELECTION CRITERIA: We included randomized controlled trials (RCTs) investigating the effectiveness of management strategies or rehabilitation approaches for structural/molecular biomarkers of knee joint health following ACL and/or meniscal tear. DATA SYNTHESIS: We included 5 RCTs (9 papers) with primary ACL tear (n = 365). Two RCTs compared initial management strategies (rehabilitation plus early vs optional delayed ACL surgery), reporting on structural biomarkers (radiographic osteoarthritis, cartilage thickness, meniscal damage) in 5 papers and molecular biomarkers (inflammation, cartilage turnover) in 1 paper. Three RCTs compared different post-ACL reconstruction (ACLR) rehabilitation approaches (high vs low intensity plyometric exercises, accelerated vs nonaccelerated rehabilitation, continuous passive vs active motion), reporting on structural biomarkers (joint space narrowing) in 1 paper and molecular biomarkers (inflammation, cartilage turnover) in 2 papers. RESULTS: There were no differences in structural or molecular biomarkers between post-ACLR rehabilitation approaches. One RCT comparing initial management strategies demonstrated that rehabilitation plus early ACLR was associated with greater patellofemoral cartilage thinning, elevated inflammatory cytokine response, and reduced incidence of medial meniscal damage over 5 years compared to rehabilitation with no/delayed ACLR. CONCLUSION: Very low-certainty evidence suggests that different initial management strategies (rehabilitation plus early vs optional delayed ACL surgery) but not postoperative rehabilitation approaches may influence the incidence of meniscal damage, patellofemoral cartilage loss and cytokine concentrations over 5 years post-ACL tear. J Orthop Sports Phys Ther 2023;53(4):1-22. Epub: 20 February 2023. doi:10.2519/jospt.2023.11576.

Biological variation of human aggrecan ARGS neoepitope in synovial fluid and serum in early-stage knee osteoarthritis and after knee injury.

Objective: To determine biological variation of the aggrecanase-generated aggrecan ARGS neoepitope in serum (sARGS) and synovial fluid (sfARGS) within and between patients with osteoarthritis (OA) or anterior cruciate ligament (ACL) injury. Design: Matched samples of serum and synovial fluid were available, as parts of clinical trials, from i) 16 subjects with early-stage OA on 8 occasions over 1 year, and ii) 120 subjects with acute ACL injury with samples available from at least 2 of 6 visits over 5 years. We used an in-house immunoassay to quantify ARGS and one-way ANOVA for statistical analyses. Results: Variability in ARGS was higher in synovial fluid than in serum in both patient groups. Subjects with OA had the lowest variability both within and between patients and showed no variation over time in the degree of variability or in the cross-sectional mean, neither in serum nor in synovial fluid. After ACL injury, the concentration and the variability of ARGS was highest immediately after injury, with a subsequent decline both in concentration and in variability with time. In both patient groups there was a positive correlation between sfARGS and sARGS both within and between individuals (correlation coefficients between 0.16 and 0.20). Conclusions: The biological variation of ARGS is lower in serum than in synovial fluid, and lower in OA than after ACL injury. Serum ARGS is a measure of the total release of ARGS aggrecan from the whole body and a poor reflection of the release of ARGS aggrecan within the affected joint.

Safety, Pharmacokinetics, and Pharmacodynamics of the ADAMTS-5 Inhibitor GLPG1972/S201086 in Healthy Volunteers and Participants With Osteoarthritis of the Knee or Hip.

GLPG1972/S201086 is a disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS-5) inhibitor in development as an osteoarthritis disease-modifying therapy. We report the safety, tolerability, pharmacokinetics, and pharmacodynamics (turnover of plasma/serum ARGS-aggrecan neoepitope fragments [ARGS]) of GLPG1972 in 3 randomized, double-blind, placebo-controlled phase 1 trials. Study A, a first-in-human trial of single (≤2100 mg [fasted] and 300 mg [fed]) and multiple (≤1050 mg once daily [fed]; 14 days) ascending oral (solution) doses, investigated GLPG1972 in healthy men (N = 41; NCT02612246). Study B investigated multiple ascending oral (tablet) doses of GLPG1972 (≤300 mg once daily [fed]; 4 weeks) in male and female participants with osteoarthritis (N = 30; NCT03311009). Study C investigated single (Japanese: ≤1500 mg; White: 300 mg [fasted]) and multiple (Japanese, ≤1050 mg once daily; White, 300 mg once daily [fed]; 14 days) ascending oral (tablet) doses of GLPG1972 in healthy Japanese and White men (N = 88). The pharmacokinetic profile of GLPG1972 was similar between healthy participants and participants with osteoarthritis, with low to moderate interindividual variability. GLPG1972 was rapidly absorbed (median time to maximum concentration, 4 hours), and eliminated with a mean apparent terminal elimination half-life of ≈10 hours. Steady state was achieved within 2 days of dosing, with minimal accumulation. Steady-state plasma exposure after 300 mg of GLPG1972 showed no or minor differences between populations. Area under the plasma concentration-time curve (56.8-67.6 µg · h/mL) and time to maximum concentration (4 hours) were similar between studies. Urinary excretion of GLPG1972 (24 hours) was low (<11%). Multiple dosing significantly reduced ARGS levels vs baseline at all time points for all doses vs placebo. GLPG1972 was generally well tolerated at all doses.

An Anterior Cruciate Ligament Rupture Increases Levels of Urine N-terminal Cross-linked Telopeptide of Type I Collagen, Urine C-terminal Cross-linked Telopeptide of Type II Collagen, Serum Aggrecan ARGS Neoepitope, and Serum Tumor Necrosis Factor-α.

BACKGROUND: An anterior cruciate ligament (ACL) rupture results in an increased risk of developing knee osteoarthritis (OA) at an early age. Before clinical signs become apparent, the OA process has already been initiated. Therefore, it is important to look at the cascade of changes, such as the activity of cytokines and proteases, which might be associated with the later development of OA. PURPOSE: To compare biomarker levels in patients with a recent ACL rupture with those in controls with a healthy knee and to monitor biomarker levels over 2 years after an ACL rupture. STUDY DESIGN: Descriptive laboratory study. METHODS: Patients were enrolled after an ACL tear was identified. Serum and urine samples were collected at the time of enrollment in the study (3-25 weeks after the injury) and then at 14 and 27 months after the injury between January 2009 and November 2010. Reference samples were obtained from participants with healthy knees. The following biomarkers were measured with immunological assays: aggrecan ARGS neoepitope (ARGS-aggrecan), tumor necrosis factor-α (TNF-α), interferon-γ, interleukin (IL)-8, IL-10, IL-13, N-terminal cross-linked telopeptide of type I collagen (NTX-I), and C-terminal cross-linked telopeptide of type II collagen (CTX-II). RESULTS: Samples were collected from 152 patients with an acute ACL rupture, who had a median age of 25 years (interquartile range [IQR], 21-32 years). There were 62 urine reference samples (median age, 25 years [IQR, 22-36 years]) and 26 serum reference samples (median age, 35 years [IQR, 24-39 years]). At a median of 11 weeks (IQR, 7-17 weeks) after trauma, serum levels of both ARGS-aggrecan and TNF-α were elevated 1.5-fold (P < .001) compared with reference samples and showed a time-dependent decrease during follow-up. Urine NTX-I and CTX-II concentrations were elevated in an early phase after trauma (1.3-fold [P < .001] and 3.7-fold [P < .001], respectively) compared with reference samples, and CTX-II levels remained elevated compared with reference samples at 2-year follow-up. Strong correlations were found between serum ARGS-aggrecan, urinary NTX-I, and urinary CTX-II (rs = 0.57-0.68). CONCLUSION: In the first few months after an ACL injury, there was a measurable increase in serum levels of ARGS-aggrecan and TNF-α as well as urine levels of NTX-I and CTX-II. These markers remained high compared with those of controls with healthy knees at 2-year follow-up.

Something Old, Something New, Something Borrowed, Something Taboo: Interaction and Creativity in Humour.

In this paper we treat humorous situations as a series of events underpinned by topoi, principles of reasoning recognised within a socio-cultural community. We claim that humorous effect in jokes and other discourse is often created by the juxtaposition of topoi evoked. A prerequisite for this is that there is a shift where the interpreter of the discourse updates their information state with regard to a second topos being evoked. This view of humour is consistent with an incremental analysis of dialogue, and we therefore argue that interaction is central both for humour creation and interpretation. We point out some different ways in which topoi are juxtaposed in humorous dialogues as well as in jokes published in social media or in joke books, and take jokes from the coronavirus pandemic as an example because this makes lots of new topoi available and therefore offers the opportunity of creating novel jokes based on the juxtaposition of the new and existing topoi. We explore how the mechanisms of inference in dialogue can be applied to humour through the four elements from our title: old (existing), new (not previously existing), borrowed (associated with a different situation) and taboo (inappropriate in the context).

Higher aggrecan 1-F21 epitope concentration in synovial fluid early after anterior cruciate ligament injury is associated with worse knee cartilage quality assessed by gadolinium enhanced magnetic resonance imaging 20 years later.

BACKGROUND: To investigate if cartilage related biomarkers in synovial fluid are associated with knee cartilage status 20 years after an anterior cruciate ligament (ACL) injury. METHODS: We studied 25 patients with a complete ACL rupture without subsequent ACL reconstruction or radiographic knee OA. All had a delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) 20 years after the ACL injury, using the T1 transverse relaxation time in the presence of gadolinium (T1Gd) which estimates the concentration of glycosaminoglycans in hyaline cartilage. Synovial fluid samples were aspirated acutely (between 0 and 18 days) and during 1 to 5 follow up visits between 0.5 and 7.5 years after injury. We quantified synovial fluid concentrations of aggrecan (epitopes 1-F21 and ARGS), cartilage oligomeric matrix protein, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by immunoassays, and sulfated glycosaminoglycans by Alcian blue precipitation. Western blot was used for qualitative analyses of aggrecan fragments in synovial fluid and cartilage samples. RESULTS: Western blot indicated that the 1-F21 epitope was located within the chondroitin sulfate 2 region of aggrecan. Linear regression analyses (adjusted for age, sex, body mass index and time between injury and sampling) showed that acute higher synovial fluid 1-F21-aggrecan concentrations were associated with shorter T1Gd values 20 years after injury, i.e. inferior cartilage quality (standardized effects between - 0.67 and - 1.0). No other statistically significant association was found between molecular biomarkers and T1Gd values. CONCLUSION: Higher acute synovial fluid 1-F21-aggrecan concentrations in ACL injured patients, who managed to cope without ACL reconstruction and were without radiographic knee OA, were associated with inferior knee cartilage quality assessed by dGEMRIC 20 years after injury.

Molecular and Structural Biomarkers of Inflammation at Two Years After Acute Anterior Cruciate Ligament Injury Do Not Predict Structural Knee Osteoarthritis at Five Years.

OBJECTIVE: To determine the role of inflammatory biomarkers at 2 years post-anterior cruciate ligament (ACL) injury to predict radiographic knee osteoarthritis (OA) and magnetic resonance imaging (MRI)-defined knee OA at 5 years postinjury, with a secondary aim of estimating the concordance of inflammatory biomarkers assessed by MRI and synovial fluid (SF) analysis. METHODS: We studied 113 patients with acute ACL injury. Knee scans using 1.5T MRIs were read for Hoffa- and effusion-synovitis. Biomarkers of inflammation that we assessed included interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor, and interferon-É£ in serum and SF, and IL-12p70 in serum. We defined the outcome as radiographic knee OA (ROA) or MRI-defined OA (MROA) at 5 years. The area under the receiver operating characteristic curve (AUC), sensitivity, and specificity were evaluated in models that included MRI features only (model 1), inflammation biomarkers only (serum [model 2a] or SF [model 2b]), both MRI features and serum biomarkers (model 3a), or both MRI features and SF (model 3b) biomarkers. Linear regression analysis was used to evaluate the association between MRI features and SF biomarkers. RESULTS: At 5 years postinjury, ROA was present in 26% of the injured knees, and MROA was present in 32%. The AUCs for ROA in each model were 0.44 (95% confidence interval [95% CI] 0.42, 0.47) for model 1, 0.62 (95% CI 0.59, 0.65) for model 2a, 0.53 (95% CI 0.50, 0.56) for model 2b, 0.58 (95% CI 0.55, 0.61) for model 3a, and 0.50 (95% CI 0.46, 0.53) for model 3b. The AUCs for MROA in each model were 0.67 (95% CI 0.64, 0.70) for model 1, 0.49 (95% CI 0.47, 0.52) for model 2a, 0.56 (95% CI 0.52, 0.59) for model 2b, 0.65 (95% CI 0.61, 0.68) for model 3a, and 0.69 (95% CI 0.66, 0.72) for model 3b. The concordance between MRI and SF biomarkers was statistically significant only for effusion-synovitis and IL-8. CONCLUSION: Neither MRI-detected inflammation nor selected SF/serum inflammation biomarkers at 2 years postinjury predicted ROA or MROA at 5 years postinjury. Concordance between MRI and SF inflammatory biomarkers was weak.

Impact of Exercise Therapy on Molecular Biomarkers Related to Cartilage and Inflammation in Individuals at Risk of, or With Established, Knee Osteoarthritis: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: To investigate the impact of exercise therapy on molecular biomarkers related to cartilage and inflammation in individuals at risk of, or with established, knee osteoarthritis by conducting a systematic review of randomized controlled trials (RCTs). METHODS: We conducted a literature search up to September 2017 in 5 major databases with no restriction on publication year or language. Data were extracted from the first available follow-up time point, and we performed a narrative synthesis for the effect of exercise therapy on molecular biomarkers related to cartilage and inflammation. A subset of studies reporting sufficient data was combined in a meta-analysis, using an adjusted random-effects model. RESULTS: Twelve RCTs involving 57 study comparisons at 4 to 24 weeks following an exercise-therapy intervention were included. Exercise therapy decreased molecular biomarkers in 17 study comparisons (30%), had no effect in 36 (63%), and increased molecular biomarkers in 4 study comparisons (7%). Meta-analyses of 9 biomarkers showed that exercise therapy was associated with nonsignificant reductions of the C-reactive protein level, C-terminal crosslinking telopeptide of type II collagen, tumor necrosis factor (TNF), soluble TNF receptors 1 and 2, C2C neoepitope of type II collagen, and cartilage oligomeric matrix protein, compared to nonexercising control groups, and exercise therapy had no effect on interleukin-6 and soluble interleukin-6 receptor. CONCLUSION: Exercise therapy is not harmful, because it does not increase the concentration of molecular biomarkers related to cartilage turnover and inflammation, implicated in osteoarthritis progression. The overall quality of evidence was downgraded to low because of the limited number of RCTs available.

α1-Microglobulin Protects Against Bleeding-Induced Oxidative Damage in Knee Arthropathies.

Knee injury increases the risk of developing knee osteoarthritis (OA). Recent evidence suggests involvement of oxidative stress induced by inflammation and bleeding in the joint. This study investigates the role in this process of α1-microglobulin (A1M), a plasma and tissue antioxidant protein with reducing function, and heme- and radical-binding properties. We studied matched knee synovial fluid (sf) and serum (s) samples from 122 subjects (mean age 40 years, 31% females): 10 were knee healthy references, 13 had acute inflammatory arthritis (AIA), 79 knee injury 0-10 years prior to sampling, and 20 knee OA. Using immunoassays, we measured sf-A1M and s-A1M, sf-hemoglobin (sf-Hb), sf-total free heme (sf-Heme), and sf-carbonyl groups (sf-Carbonyl). We explored associations by partial correlation, or linear regression models with adjustments for age, sex and diagnosis, and evaluated diagnostic capacity by area under the receiver operator characteristics curve (AUC). The AIA group had 1.2- to 1.7-fold higher sf-A1M and s-A1M concentrations compared to the other diagnostic groups; other biomarkers showed no between-group differences. sf-A1M and s-A1M were with AUC of 0.76 and 0.78, respectively, diagnostic for AIA. In the injury group, the amount of bleeding in the joint was inversely correlated to time after injury when measured as sf-Heme (r = -0.41, p < 0.001), but not when measured as sf-Hb (r = -0.19, p = 0.098). A similar inverse association with time after injury was noted for sf-A1M (r = -0.30, p = 0.007), but not for s-A1M and sf-Carbonyl. Linear regression models showed that sf-Heme was more strongly associated with sf-A1M and sf-Carbonyl than sf-Hb. Independent of diagnosis, sf-Heme explained 5.7% of the variability in sf-A1M and 3.0% in the variability in sf-Carbonyl, but appeared unrelated to s-A1M. High sf-A1M and low sf-Heme or sf-Hb were independently associated with low sf-Carbonyl. In conclusion, our results demonstrate that independent of disease, Hb and heme within a knee joint correlates with an increased sf-A1M concentration that appears to be protective of oxidative damage, i.e., a reduction in carbonyl groups. High concentrations of A1M in synovial fluid and serum was further diagnostic for AIA.

Time between anterior cruciate ligament injury and reconstruction and cartilage metabolism six-months following reconstruction.

BACKGROUND: To determine the association between time from injury to ACL reconstruction (TimeInjury-ACLR) and biochemical markers of cartilage metabolism and inflammation six months following ACL reconstruction (ACLR). METHODS: Individuals with a unilateral ACL injury were enrolled at initial presentation in the orthopedic clinic; blood was collected six months following ACLR. Enzyme-linked immunosorbent assays were used to analyze the ratio of serum concentrations of type-II collagen breakdown (C2C) to synthesis (CPII), plasma matrix metalloproteinase-3 (MMP-3), interleukin-6 (IL-6), and serum aggrecan neoepitope (ARGS). We used separate linear regressions to assess associations between biochemical markers and TimeInjury-ACLR. RESULTS: Twenty-two participants (50% females, mean [SD], age 21.9 [4.5] years old; BMI 23.8 [2.6] kg/m2) completed the study. TimeInjury-ACLR ranged from nine to 67days (31.0 [14.4days]). Greater TimeInjury-ACLR predicted greater serum C2C:CPII ratios six months following ACLR (C2C:CPII=0.15 [0.02], R2=0.213, P=0.030). Males (R2=0.733, P=0.001) but not females (R2=0.030, P=0.609) demonstrated a significant association between greater C2C:CPII and TimeInjury-ACLR at the six-month follow-up exam. TimeInjury-ACLR did not associate with IL-6, MMP-3, or ARGS at six months. CONCLUSIONS: Greater time between injury and ACL reconstruction was associated with greater serum C2C:CPII six months following ACLR in males but not females, and IL-6, MMP-3, and ARGS levels were not associated with TimeInjury-ACLR in males or females. The time between ACL injury and ACLR may affect collagen metabolism in males and should be further investigated in a larger study along with other patient-relevant outcomes.

Grounding as a Side-Effect of Grounding.

In relation to semantics, "grounding" has (at least) two relevant meanings. "Symbol grounding" is the process of connecting symbols (e.g., words) to perception and the world. "Communicative grounding" is the process of interactively adding to common ground in dialog. Strategies for grounding in human communication include, crucially, strategies for resolving troubles caused by various kinds of miscommunication. As it happens, these two processes of grounding are closely related. As a side-effect of grounding an utterance, dialog participants (DPs) may adjust the meanings they assign to linguistic expressions, in a process of semantic coordination. Meanings of at least some expressions (e.g., concrete nouns) include perceptual aspects which enable DPs to classify entities as falling under the expression or not based on their perception of those entities. We show how perceptual grounding of symbols can be achieved in a process of interactively adding to common ground. This requires that perceptual aspects of meaning can be updated as a result of participating in linguistic interaction, thereby enabling fine-grained semantic coordination of perceptually grounded linguistic meanings. A formal semantics for low-level perceptual aspects of meaning is presented, tying these together with the logical-inferential aspects of meaning traditionally studied in formal semantics. The key idea is to model perceptual meanings as classifiers of perceptual input. This requires a framework where intensions are (a) represented independently of extensions, and (b) structured objects which can be modified as a result of learning. We use Type Theory with Records (TTR), a formal semantics framework which starts from the idea that information and meaning are founded on our ability to perceive and classify the world, that is, to perceive objects and situations as being of types. As an example of our approach, we show how a simple classifier of spatial information based on the Perceptron can be cast in TTR.

Fast-track access to urologic care for patients with macroscopic haematuria is efficient and cost-effective: results from a prospective intervention study.

BACKGROUND: The delay between onset of macroscopic haematuria and diagnosis of bladder cancer is often long. METHODS: We evaluated timely diagnosis and health-care costs for patients with macroscopic haematuria given fast-track access to diagnostics. During a 15-month period, a telephone hotline for fast-track diagnostics was provided in nine Swedish municipalities for patients aged ⩾50 years with macroscopic haematuria. The control group comprised 101 patients diagnosed with bladder cancer in the same catchment area with macroscopic haematuria who underwent regular diagnostic process. RESULTS: In all 275 patients who called 'the Red Phone' hotline were investigated, and 47 of them (17%) were diagnosed with cancer and 36 of those had bladder cancer. Median time from patient-reported haematuria to diagnosis was 29 (interquartile range (IQR) 14-104) days and 50 (IQR 27-165) days in the intervention and the control group, respectively (P=0.03). The median health-care costs were lower in the intervention group (655 (IQR 655-655) EUR) than in the control group (767 (IQR 490-1096) EUR) (P=0.002). CONCLUSIONS: Direct access to urologic expertise and fast-track diagnostics is motivated for patients with macroscopic haematuria to reduce diagnostic intervals and lower health-care expenditures.

Changes in Cytokines and Aggrecan ARGS Neoepitope in Synovial Fluid and Serum and in C-Terminal Crosslinking Telopeptide of Type II Collagen and N-Terminal Crosslinking Telopeptide of Type I Collagen in Urine Over Five Years After Anterior Cruciate Ligament Rupture: An Exploratory Analysis in the Knee Anterior Cruciate Ligament, Nonsurgical Versus Surgical Treatment Trial.

OBJECTIVE: To prospectively monitor levels of proinflammatory cytokines and aggrecan ARGS neoepitope in synovial fluid and serum as well as levels of C-terminal crosslinking telopeptide of type II collagen (CTX-II) and N-terminal crosslinking telopeptide of type I collagen (NTX-I) in urine after acute anterior cruciate ligament (ACL) rupture. METHODS: Synovial fluid, serum, and urine were collected from 121 adults on 6 occasions over 5 years after acute ACL injury. Reference samples were obtained from subjects without knee injury. Concentrations of interleukin-6 (IL-6), IL-8, IL-10, interferon-γ (IFNγ), tumor necrosis factor (TNF), aggrecan ARGS neoepitope, CTX-II, and NTX-I were measured by enzyme-linked immunosorbent assay. RESULTS: Shortly after ACL injury, cytokine concentrations in synovial fluid were elevated 6-fold (TNF) to 1,050-fold (IL-6) compared to reference levels, while concentrations of aggrecan ARGS neoepitope in synovial fluid and serum and CTX-II in urine were elevated 1.4-fold to 8-fold. Thereafter, concentrations of cytokines and aggrecan ARGS neoepitope in synovial fluid decreased with different half-lives (in years: IL-6 0.9, IL-8 2.2, IL-10 2.3, IFNγ 3.1, TNF 3.6, aggrecan ARGS neoepitope 4.0). After 5 years, the TNF concentration in synovial fluid remained higher than the reference level. There was a correlation between the concentrations of aggrecan ARGS neoepitope in synovial fluid and serum (rs = 0.36). Concentrations of aggrecan ARGS neoepitope in synovial fluid and of CTX-II and NTX-I in urine were correlated with concentrations of cytokines in synovial fluid (rs = 0.41-0.49 and rs = 0.21-0.31, respectively). CONCLUSION: Acute ACL injury induced highly increased levels of inflammatory cytokines in the joint, and these were associated with proteolysis of aggrecan and type II collagen. Cytokine levels remained increased up to 5 years after injury, indicative of extended local inflammation in the joint.

A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid.

In the study of aggrecan fragmentation several methods to extract and purify aggrecan from cartilage and synovial fluid (SF) are used. This work compares and evaluates the effectiveness for purification of aggrecan of the most commonly used methods by the ratio of sulfated glycosaminoglycan (sGAG) to protein and by fragment analysis by Western blot. A novel method for purification of aggrecan fragments from SF by boiling (Boiled SF) is also presented. Of the sGAG extracted from cartilage by guanidinium, 66% was recovered by associative-dissociative cesium chloride density gradient centrifugation (A1D1-D3) with a 9 times higher ratio of sGAG to protein in the A1D1 fraction. Although less enriched in aggrecan, the Western blot aggrecan pattern of the guanidinium extracted sample resembled that of the combined patterns of the A1D1, A1D2 and A1D3 fractions. The recoveries of sGAG from SF purified by anion chromatography and Alcian blue precipitation were around 50%, while the recoveries were over 80% in the associative or dissociative density gradient fractions (A1 and D1) and Boiled SF. The purification compared to neat SF ranged from 9 times in boiled SF to 1800-1900 times in Alcian blue and D1 samples. To obtain reliable results when analyzing synovial fluid aggrecan fragments by Western blot, purification was necessary. The immuno-pattern of anion chromatography purified SF resembled the patterns of A1 and D1, while the pattern of Boiled SF resembled the D1 sample. This work suggests that aggrecan fragments extracted from cartilage by guanidinium need no further purification to be analyzed by Western blot, whereas aggrecan fragments in SF are best analyzed in the A1 and D1 fractions or in the Boiled SF sample.

Association between synovial fluid levels of aggrecan ARGS fragments and radiographic progression in knee osteoarthritis.

INTRODUCTION: Aggrecanase cleavage at the (392)Glu-(393)Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal (393)ARGS fragments, is an early key event in arthritis and joint injuries. We determined whether synovial fluid (SF) levels of ARGS-aggrecan distinguish subjects with progressive radiographic knee osteoarthritis (ROA) from those with stable or no ROA. METHODS: We studied 141 subjects who, at examination A, had been given meniscectomies an average of 18 years earlier (range, 15 to 22 years). Seventeen individuals without surgery, and without known injury to the menisci or cruciate ligaments, were used as references. At examinations A and B, with a mean follow-up time of 7.5 years, we obtained SF and standing tibiofemoral and skyline patellofemoral radiographs. SF ARGS-aggrecan was measured with an electrochemiluminescence immunoassay, and we graded radiographs according to the OARSI atlas. The association between SF ARGS levels at examination A and progression of radiographic features of knee OA between examinations A and B was assessed by using logistic regression adjusted for age, gender, body mass index, and time between examinations, and stratified by ROA status at examination A. RESULTS: We found a weak negative association between SF ARGS concentrations and loss of joint space: the likelihood of progression of radiographic joint space narrowing decreased 0.9 times per picomole per milliliter increase in ARGS (odds ratio (OR) 0.89; 95% confidence interval (CI), 0.79 to 0.996). In subjects with and without preexisting ROA at examination A, the association was OR, 0.96; 0.81 to 1.13; and 0.77; 0.62 to 0.95, respectively. Average levels of SF ARGS 18 years after meniscectomy were no different from those of reference subjects and were not correlated to radiographic status at examination A. CONCLUSIONS: In subjects with previous knee meniscectomy but without ROA, levels of SF ARGS-aggrecan were weakly and inversely associated with increased loss of joint space over a period of 7.5 years.

Synovial fluid level of aggrecan ARGS fragments is a more sensitive marker of joint disease than glycosaminoglycan or aggrecan levels: a cross-sectional study.

INTRODUCTION: Aggrecanase cleavage at the 392Glu-393Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal 393ARGS fragments, is an early key event in arthritis and joint injuries. Here, we use a quantitative immunoassay of aggrecan ARGS neoepitope fragments in human synovial fluid to determine if this cleavage-site specific method better identifies joint pathology than previously available less specific aggrecan assays. METHODS: Synovial fluid (SF) from 26 people with healthy knees (reference) and 269 patients were analyzed in a cross-sectional study. Patient groups were acute inflammatory arthritis, acute knee injury, chronic knee injury and knee osteoarthritis (OA). Aggrecan ARGS fragments were assayed by ELISA using the monoclonal antibody OA-1. Total aggrecan content was analyzed by an ELISA using the monoclonal antibody 1-F21, and sulfated glycosaminoglycan by Alcian blue precipitation. RESULTS: Aggrecan ARGS fragment concentrations in all groups differed from the reference group (P < 0.001). The acute inflammatory arthritis group had the highest median level, 177-fold greater than that of the reference group. Median levels (in pmol ARGS/ml SF) were: reference 0.5, acute inflammatory arthritis 88.5, acute knee injury 53.9, chronic knee injury 0.5 and OA 4.6. In contrast, aggrecan and sulfated glycosaminoglycan concentrations varied much less between groups, and only acute inflammatory arthritis and acute knee injury were found to have a two-fold increase in median levels compared to the reference. CONCLUSIONS: Levels of aggrecan ARGS fragments in human synovial fluid are increased in human arthritis, OA and after knee injury, likely reflecting an enhanced cleavage at the 392Glu-393Ala bond in the IGD by aggrecanase. An assay that specifically quantified these fragments better distinguished samples from joints with pathology than assays monitoring aggrecan or glycosaminoglycan concentrations. The newly developed ARGS fragment assay can be used to monitor aggrecanase activity in human joint disease and experimental models.

Use of the plasma stromelysin (matrix metalloproteinase 3) concentration to predict joint space narrowing in knee osteoarthritis.

OBJECTIVE: To determine whether baseline or serial plasma concentrations of stromelysin (matrix metalloproteinase 3 [MMP-3]) protein might distinguish subjects with progressive radiographic knee osteoarthritis (OA) from those with stable disease. METHODS: Subjects were 120 women with unilateral knee OA who participated in a 30-month randomized, placebo-controlled trial of structure modification with doxycycline. Anteroposterior views of both knees in a semiflexed position were obtained at baseline, 16 months, and 30 months. Subjects were selected to obtain comparisons of plasma MMP-3 levels between 60 progressors (21 taking doxycycline, 39 taking placebo) and 60 nonprogressors (30 taking doxycycline, 30 taking placebo) with respect to medial joint space narrowing (JSN) in the index knee. Each group consisted of 30 subjects who exhibited significant increases in knee pain. Blood samples were obtained semiannually for MMP-3 assay. RESULTS: Subjects in the placebo group whose MMP-3 concentration was in the upper tertile of the baseline distribution showed a 4-fold increase in the odds of progression of JSN as compared with the lower tertile (odds ratio 4.12, P = 0.037). Baseline MMP-3 levels were unrelated to knee pain. The within-subject mean of serial MMP-3 concentrations was associated with concurrent JSN in the placebo group over the 0-16-month interval (b = 0.18 mm/SD increase in the mean MMP-3, P < 0.01) and over the 16-30-month interval (b = 0.15, P < 0.05). Similar evidence of concurrent validity was found in the placebo group for the maximum of intercurrent MMP-3 values. CONCLUSION: The baseline MMP-3 level was a significant predictor of JSN in this pilot study. Moreover, serial plasma MMP-3 levels reflected concurrent JSN in the placebo group over the 30-month period of observation.

Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells.

The cytokine tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. Through its ability to promote ex vivo expansion and oncoretroviral transduction of primitive human hematopoietic progenitors, the flt3 ligand (FL) has emerged as a key stimulator of candidate human hematopoietic stem cells (HSCs). However, recent studies in the mouse suggest that though it is present on short-term repopulating cells, flt3 is not expressed on bone marrow long-term reconstituting HSCs, the ultimate target for the development of cell replacement and gene therapy. Herein we demonstrate that though only a fraction of human adult bone marrow and cord blood CD34+long-term culture-initiating cells (LTC-ICs) express flt3, most cord blood lymphomyeloid HSCs capable of in vivo reconstituting nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice are flt3+. The striking difference in flt3 and c-kit expression on mouse and candidate human HSCs translated into a corresponding difference in flt3 and c-kit function because FL was more efficient than SCF at supporting the survival of candidate human HSCs. In contrast, SCF is far superior to FL as a viability factor for mouse HSCs. Thus, the present data provide compelling evidence for a contrasting expression and response pattern of flt3 and c-kit on mouse and human HSCs.