Generation of Bayesian prediction models for OATP-mediated drug-drug interactions based on inhibition screen of OATP1B1, OATP1B1∗15 and OATP1B3.

Human organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are important hepatic uptake transporters. Early assessment of OATP1B1/1B3-mediated drug-drug interactions (DDIs) is therefore important for successful drug development. A promising approach for early screening and prediction of DDIs is computational modeling. In this study we aimed to generate a rapid, single Bayesian prediction model for OATP1B1, OATP1B1∗15 and OATP1B3 inhibition. Besides our previously generated HEK-OATP1B1 and HEK-OATP1B1∗15 cells, we now generated and characterized HEK-OATP1B3 cells. Using these cell lines we investigated the inhibitory potential of 640 FDA-approved drugs from a commercial library (10µM) on the uptake of [(3)H]-estradiol-17ß-d-glucuronide (1µM) by OATP1B1, OATP1B1∗15, and OATP1B3. Using a cut-off of ⩾60% inhibition, 8% and 7% of the 640 drugs were potent OATP1B1 and OATP1B1∗15 inhibitors, respectively. Only 1% of the tested drugs significantly inhibited OATP1B3, which was not sufficient for Bayesian modeling. Modeling of OATP1B1 and OATP1B1∗15 inhibition revealed that presence of conjugated systems and (hetero)cycles with acceptor/donor atoms in- or outside the ring enhance the probability of a molecule binding these transporters. The overall performance of the model for OATP1B1 and OATP1B1∗15 was ⩾80%, including evaluation with a true external test set. Our Bayesian classification model thus represents a fast, inexpensive and robust means of assessing potential binding of new chemical entities to OATP1B1 and OATP1B1∗15. As such, this model may be used to rank compounds early in the drug development process, helping to avoid adverse effects in a later stage due to inhibition of OATP1B1 and/or OATP1B1∗15.

Canadian parents' attitudes and beliefs about bicycle helmet legislation in provinces with and without legislation.

INTRODUCTION: The objective of this study was to survey Canadian parents on their attitudes and beliefs about bicycle helmet legislation and to compare responses from parents living in provinces with and without legislation. METHODS: A national survey of 1002 parents of children aged under 18 years was conducted. Chi-square tests were used to compare responses from the surveyed parents in the different jurisdictions. RESULTS: Responses from parents living in provinces with legislation (n = 640) and without legislation (n = 362) were as follows: concern for injury (63% vs. 68%, nonsignificant [NS]); believe helmets are effective (98% vs. 98%, NS); child always wears a helmet (74% vs. 69%, NS); support legislation for children (95% vs. 83%, p < .001); support legislation for all ages (85% vs. 75%, p < .001); support police enforcement (83% vs. 76%, p = .003); believe legislation decreases the amount of time their child bicycles (5% vs. 8%, NS). CONCLUSION: Parents are highly supportive of bicycle helmet legislation in Canada. They believe that bicycle helmets are effective and that legislation does not decrease the amount of time a child spends bicycling. There was also a high level of support for legislation across all ages, and for police enforcement.

TITRE: Attitudes et croyances des parents au sujet du port obligatoire du casque de vélo : comparaison entre provinces avec et sans législation. INTRODUCTION: L'objectif de l'étude était d'effectuer un sondage auprès de parents canadiens sur leurs attitudes et leurs croyances au sujet de la législation du port du casque chez les cyclistes et de comparer les réponses des parents vivant dans une province ayant légiféré dans ce domaine et celles de ceux vivant dans une province n'ayant pas légiféré. MÉTHODOLOGIE: Un sondage national a été réalisé auprès de 1 002 parents d'enfants âgés de moins de 18 ans. Des tests du chi-carré ont été utilisés pour comparer les réponses des parents dans les différentes provinces. RÉSULTATS: Les réponses des parents vivant dans une province ayant légiféré (n = 640) ou ne l'ayant pas fait (n = 362) s'établissent respectivement comme suit : sont préoccupés par les accidents de vélo (63 % et 68 %, non significatif [NS]); croient que le port du casque de vélo est une mesure efficace (98 % et 98 %, NS); l'enfant porte toujours un casque de vélo (74 % et 69 %, NS); sont favorables à une loi visant les enfants (95 % et 83 %, p < 0,001); sont favorables à une loi visant les cyclistes de tous âges (85 % et 75 %, p < 0,001); sont favorables à l'application de la loi par les services de police (83 % et 76 %, p = 0,003); croient que la loi réduit la durée d'utilisation de la bicyclette par leur enfant (5 % et 8 %, NS). CONCLUSION: Les parents sont largement favorables à la législation du port du casque chez les cyclistes au Canada. Ils croient que le casque de vélo est une mesure efficace et que la loi n'a pas pour effet de réduire la durée d'utilisation de la bicyclette par leur enfant. Ils sont également largement favorables à une loi visant les cyclistes de tous âges et à l'application de la loi par les services de police.

Combined analysis of pharmacokinetic and efficacy data of preclinical studies with statins markedly improves translation of drug efficacy to human trials.

Correct prediction of human pharmacokinetics (PK) and the safety and efficacy of novel compounds based on preclinical data, is essential but often fails. In the current study, we aimed to improve the predictive value of ApoE*3Leiden (E3L) transgenic mice regarding the cholesterol-lowering efficacy of various statins in humans by combining pharmacokinetic with efficacy data. The efficacy of five currently marketed statins (atorvastatin, simvastatin, lovastatin, pravastatin, and rosuvastatin) in hypercholesterolemic patients (low-density lipoprotein ≥ 160 mg/dl) was ranked based on meta-analysis of published human trials. Additionally, a preclinical combined PK efficacy data set for these five statins was established in E3L mice that were fed a high-cholesterol diet for 4 weeks, followed by 6 weeks of drug intervention in which statins were supplemented to the diet. Plasma and tissue levels of the statins were determined on administration of (radiolabeled) drugs (10 mg/kg p.o.). As expected, all statins reduced plasma cholesterol in the preclinical model, but a direct correlation between cholesterol lowering efficacy of the different statins in mice and in humans did not reach statistical significance (R(2) = 0.11, P < 0.57). It is noteworthy that, when murine data were corrected for effective liver uptake of the different statins, the correlation markedly increased (R(2) = 0.89, P < 0.05). Here we show for the first time that hepatic uptake of statins is related to their cholesterol-lowering efficacy and provide evidence that combined PK and efficacy studies can substantially improve the translational value of the E3L mouse model in the case of statin treatment. This strategy may also be applicable for other classes of drugs and other preclinical models.

Skin pentosidine in very early hip/knee osteoarthritis (CHECK) is not a strong independent predictor of radiographic progression over 5 years follow-up.

OBJECTIVES: Age-related changes in articular cartilage are likely to play a role in the etiology of osteoarthritis (OA). One of the major age-related changes in cartilage is the accumulation of advanced glycation end products (AGEs). The present study evaluates whether pentosidine can predict radiographic progression and/or burden over 5 years follow-up in a cohort of early knee and/or hip OA. DESIGN: The 5 years follow-up data of 300 patients from cohort hip & cohort knee (CHECK) were used. Radiographic progression and burden were assessed by X-rays of both knees and hips (Kellgren and Lawrence (K&L) and Altman scores). Baseline pentosidine levels (and urinary CTXII as a comparator) were measured by high-performance-liquid-chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA). Univariable and multivariable associations including baseline radiographic damage, age, gender, body mass index (BMI) and kidney function were performed. RESULTS: Both pentosidine and urinary C-terminal telopeptide of type II collagen (uCTXII) correlated with radiographic progression and burden. In general pentosidine did not have an added predictive value to uCTXII for progression nor burden of the disease. The best prediction was obtained for burden of radiographic damage (R(2) = 0.60-0.88), bus this was predominantly determined by baseline radiographic damage (without this parameter R(2) = 0.07-0.17). Interestingly, pentosidine significantly added to prediction of osteophyte formation, whereas uCTXII significantly added to prediction of JSN in multivariable analysis. CONCLUSION: Pentosidine adds to prediction of radiographic progression and burden of osteophyte formation and uCTXII to radiographic progression and burden of JSN, but overall skin pentosidine did not perform better that uCTXII in predicting radiographic progression or burden. Burden of damage over 5 years is mainly determined by radiographic joint damage at baseline.

Drug-drug interactions between rosuvastatin and oral antidiabetic drugs occurring at the level of OATP1B1.

Organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter, of which the polymorphic variant OATP1B1*15 (Asn130Asp and Val174Ala) has been associated with decreased transport activity. Rosuvastatin is an OATP1B1 substrate and often concomitantly prescribed with oral antidiabetics in the clinic. The aim of this study was to investigate possible drug-drug interactions between these drugs at the level of OATP1B1 and OATP1B1*15. We generated human embryonic kidney (HEK)293 cells stably overexpressing OATP1B1 or OATP1B1*15 that showed similar protein expression levels of OATP1B1 and OATP1B1*15 at the cell membrane as measured by liquid chromatography-tandem mass spectrometry. In HEK-OATP1B1*15 cells, the V(max) for OATP1B1-mediated transport of E(2)17ß-G (estradiol 17ß-d-glucuronide) was decreased >60%, whereas K(m) values (Michaelis constant) were comparable. Uptake of rosuvastatin in HEK-OATP1B1 cells (K(m) 13.1 ± 0.43 µM) was nearly absent in HEK-OATP1B1*15 cells. Interestingly, several oral antidiabetics (glyburide, glimepiride, troglitazone, pioglitazone, glipizide, gliclazide, and tolbutamide), but not metformin, were identified as significant inhibitors of the OATP1B1-mediated transport of rosuvastatin. The IC(50) values for inhibition of E(2)17ß-G uptake were similar between OATP1B1 and OATP1B1*15. In conclusion, these studies indicate that several oral antidiabetic drugs affect the OATP1B1-mediated uptake of rosuvastatin in vitro. The next step will be to translate these data to the clinical situation, as it remains to be established whether the studied oral antidiabetics indeed affect the clinical pharmacokinetic profile of rosuvastatin in patients.

Clusters within a wide spectrum of biochemical markers for osteoarthritis: data from CHECK, a large cohort of individuals with very early symptomatic osteoarthritis.

OBJECTIVE: To assess a wide spectrum of biochemical markers (biomarkers) in a large cohort of individuals with (very) early symptomatic knee and/or hip osteoarthritis (OA). Secondly, to investigate associations between biomarkers and between biomarkers and demographics to demonstrate validity of the obtained dataset and further investigate the involvement and/or role of these biomarkers in OA. DESIGN: Fourteen biomarkers (uCTX-II, uCTX-I, uNTX-I, sCOMP, sPIIANP, sCS846, sC1,2C, sOC, sPINP, sHA, sPIIINP, pLeptin, pAdiponectin, pResistin) were assessed by ELISA or RIA in CHECK (Cohort Hip and Cohort Knee), a 10-year prospective cohort of 1,002 individuals with early symptomatic knee and/or hip OA. RESULTS: Quality controls revealed that gathered data were technically reliable. The majority of biomarkers showed relevant associations with demographic variables, which were expectedly different between genders and/or menopausal status for some. Principal component analysis enabled identification of five clusters, consecutively designated as 'bone-CTX-II', 'inflammation', 'synovium', 'C1,2C-adipokines', and 'cartilage synthesis' cluster. Notably, uCTX-II clustered with biomarkers of bone metabolism, while sCOMP clustered with biomarkers of synovial activity. CONCLUSIONS: The identified clusters extended knowledge on individual biomarkers from mostly smaller studies as did the observed associations between biomarker levels and demographics, from which validity of our data was deduced. uCTX-II may not only reflect articular cartilage but also bone metabolism and sCOMP may reflect synovial rather than cartilage metabolism. Major involvement of adipokines in joint metabolism was not identified.

In end stage osteoarthritis, cartilage tissue pentosidine levels are inversely related to parameters of cartilage damage.

OBJECTIVES: Age is the most prominent predisposition for development of osteoarthritis (OA). Age-related changes of articular cartilage are likely to play a role. Advanced glycation endproducts (AGEs) accumulate in cartilage matrix with increasing age and adversely affect the biomechanical properties of the cartilage matrix and influence chondrocyte activity. In clinical studies AGEing of cartilage and its relation to actual cartilage damage can only be measured by surrogate markers (e.g., serum, skin or urine AGE levels and imaging or biochemical markers of cartilage damage). In this study actual cartilage AGE levels were directly related to actual cartilage damage by use of cartilage obtained at joint replacement surgery. METHODS: Cartilage and urine samples were obtained from 69 patients undergoing total knee replacement. Samples were analyzed for pentosidine as marker of AGE. Cartilage damage was evaluated macroscopically, histologically, and biochemically. RESULTS: Cartilage and urine pentosidine both increased with increasing age. The higher the macroscopic, histological, and biochemical cartilage damage the lower the cartilage pentosidine levels were. In multiple regression analysis age is not found to be a confounder. CONCLUSION: There is an inverse relation between cartilage AGEs and actual cartilage damage in end-stage OA. This is likely due to ongoing (ineffective) increased turnover of cartilage matrix proteins even in end stage disease.

Patterns of fatal machine rollovers in Canadian agriculture.

INTRODUCTION: Our objectives were to examine the activities and circumstances associated with agricultural machine-related rollover fatalities. METHODS: We identified agricultural machine rollover fatalities recorded by the Canadian Agricultural Injury Surveillance Program (CAISP) in 1990-2005. We determined sideways and backwards rollovers by year, age and sex of the victims, agricultural season, machine type, and the activity, circumstances and location of the injury event. RESULTS: The annual rate of rollover fatalities in Canada was 9.1 per 100,000 farm operations. Rollover fatalities decreased to 30% of baseline over the 16-year study period (p = .004). Fatal rollovers most often occurred among men aged 50-69 years and 60-79 years for sideways and backwards rollovers, respectively. DISCUSSION: Sideways rollovers occur when driving across an incline or at the edge of a ditch bordering a roadway or field. Backwards rollovers occur when driving up an incline, towing or extracting stuck machines, pulling stumps or trees, and towing implements or logs. Primary prevention programs for rollover injuries should target these identified patterns of injury.

The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the guinea pig.

OBJECTIVE: This review focuses on the criteria for assessing osteoarthritis (OA) in the guinea pig at the macroscopic and microscopic levels, and recommends particular assessment criteria to assist standardization in the conduct and reporting of preclinical trails in guinea pig models of OA. METHODS: A review was conducted of all OA studies from 1958 until the present that utilized the guinea pig. The PubMed database was originally searched August 1, 2006 using the following search terms: guinea pig and OA. We continued to check the database periodically throughout the process of preparing this chapter and the final search was conducted January 7, 2009. Additional studies were found in a review of abstracts from the OsteoArthritis Research Society International (OARSI) conferences, Orthopaedic Research Society (ORS) conferences, and literature related to histology in other preclinical models of OA reviewed for relevant references. Studies that described or used systems for guinea pig joint scoring on a macroscopic, microscopic, or ultrastructural basis were included in the final comprehensive summary and review. General recommendations regarding methods of OA assessment in the guinea pig were derived on the basis of a comparison across studies and an inter-rater reliability assessment of the recommended scoring system. RESULTS: A histochemical-histological scoring system (based on one first introduced by H. Mankin) is recommended for semi-quantitative histological assessment of OA in the guinea pig, due to its already widespread adoption, ease of use, similarity to scoring systems used for OA in humans, its achievable high inter-rater reliability, and its demonstrated correlation with synovial fluid biomarker concentrations. Specific recommendations are also provided for histological scoring of synovitis and scoring of macroscopic lesions of OA. CONCLUSIONS: As summarized herein, a wealth of tools exist to aid both in the semi-quantitative and quantitative assessment of OA in the guinea pig and provide a means of comprehensively characterizing the whole joint organ. In an ongoing effort at standardization, we recommend specific criteria for assessing the guinea pig model of OA as part of an OARSI initiative, termed herein the OARSI-HISTOgp recommendations.

Skin and urine pentosidine weakly correlate with joint damage in a cohort of patients with early signs of osteoarthritis (CHECK).

OBJECTIVES: Age-related changes in articular cartilage are likely to play a role in the aetiology of osteoarthritis (OA). One of the major age-related changes in cartilage is the accumulation of advanced-glycation-endproducts (AGEs). Since, cartilage tissue is not readily available from patients for studying AGE levels, alternative approaches such as analyzing skin and urine are needed to study the role of cartilage AGE levels in OA. METHODS: Paired human skin and cartilage samples were obtained post mortem. Paired skin and urine samples were obtained from the CHECK cohort (early OA patients). Pentosidine levels were measured by high-performance liquid chromatography (HPLC). As marker of cumulative cartilage damage X-rays of both knees and hips were scored. Urinary CTXII (uCTXII) levels were measured, to assess current cartilage breakdown. RESULTS: Cartilage and skin pentosidine correlate well (R=0.473, P=0.05). Skin pentosidine was higher in mild (summed (Kellgren & Lawrence K&L) over four large joints ≥4) compared to no (summed K&L≤3) radiographic OA (P=0.007). Urinary pentosidine was not different between these two groups. Skin pentosidine levels were not related to cartilage breakdown (highest vs lowest tertile of uCTXII). Urinary pentosidine, however, was higher in the highest compared to the lowest uCTXII tertile (P=0.009). Multiple regression analysis showed age to be the only predictor of the summed K&L score and age, creatinine clearance and urinary pentosidine as predictors of uCTXII. CONCLUSION: The higher skin and urinary pentosidine levels in those with mild compared to no radiographic joint damage and low vs high cartilage breakdown respectively suggest that AGEs may contribute to disease susceptibility and/or progression. However, relations are weak and cannot be used as surrogate markers of severity of OA.

Serum and urinary biochemical markers for knee and hip-osteoarthritis: a systematic review applying the consensus BIPED criteria.

CONTEXT: Molecules that are released into biological fluids during matrix metabolism of articular cartilage, subchondral bone, and synovial tissue could serve as biochemical markers of the process of osteoarthritis (OA). Unfortunately, actual breakthroughs in the biochemical OA marker field are limited so far. OBJECTIVE: By reviewing the status of commercially available biochemical OA markers according to the "Burden of disease, Investigative, Prognostic, Efficacy of intervention, and Diagnostic" ("BIPED") classification, future use of this "BIPED" classification is encouraged and more efficient biochemical OA marker research stimulated. DATA SOURCES: Three electronic databases [PubMed, Scopus, EMBASE (1997-May 2009)] were searched for publications on blood and urinary biochemical markers in human primary knee and hip-OA. STUDY SELECTION: Stepwise selection of original English publications describing human studies on blood or urinary biochemical markers in primary knee or hip-OA was performed. Selected articles were fully read to determine whether biochemical markers were investigated on performance within any of the "BIPED" categories. Eighty-four relevant publications were identified. DATA EXTRACTION: Data from relevant publications were tabulated according to the "BIPED" classification. Individual analyses within a publication were summarized in general "BIPED" scores. DATA SYNTHESIS: An uneven distribution of scores on biochemical marker performance and heterogeneity among the publications complicated direct comparison of individual biochemical markers. Comparison of categories of biochemical markers was therefore performed instead. In general, biochemical markers of cartilage degradation were investigated most extensively and performed well in comparison with other categories of biochemical markers. Biochemical markers of bone metabolism performed less adequately. Biochemical markers of synovial tissue metabolism were not investigated extensively, but performed quite well. CONCLUSIONS: Specific biochemical markers and categories of biochemical markers as well as their nature, origin and metabolism, need further investigation. International standardization of future investigations should be pursued to obtain more high-quality, homogenous data on the full spectrum of biochemical OA markers.

Elevated Levels of Cartilage Oligomeric Matrix Protein during In Vitro Cartilage Matrix Generation Decrease Collagen Fibril Diameter.

Cartilage oligomeric matrix protein (COMP) is a protein present in the cartilage matrix and is expressed more abundantly in osteoarthritis cartilage than in healthy cartilage. The present study was designed to investigate the effect of transforming growth factor ß (TGFß) on COMP deposition and the influence of COMP on collagen biochemistry in a long-term 3-dimensional culture. Bovine chondrocytes in alginate beads were cultured with or without 25 ng/mL TGFß2 for 21 or 35 days. COMP was overexpressed in bovine chondrocytes using lentiviral transfection. COMP gene expression, COMP protein production, collagen and proteoglycan deposition, and collagen fibril thickness were determined. Addition of TGFß2 resulted in more COMP mRNA and protein than the control condition without growth factors. Lentiviral transduction with COMP resulted in elevated gene expression of COMP and increased COMP levels in the alginate bead and culture medium compared to untransfected cells. Overexpression of COMP did not affect the deposition of collagen, collagen cross-linking, proteoglycan deposition, or the mechanical properties. Stimulating COMP production by either TGFß2 or lentivirus resulted in collagen fibrils with a smaller diameter. Taken together, COMP deposition can be modulated in cartilage matrix production by the addition of growth factors or by overexpression of COMP. Inducing COMP protein expression resulted in collagen fibrils with a smaller diameter. Because it has been demonstrated that the collagen fibril diameter is associated with mechanical functioning of the matrix, modulating COMP levels should be taken into account in cartilage regeneration strategies.

Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: cartilage oligomeric matrix protein counteracts type IX collagen-induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures.

OBJECTIVE: Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins type IX collagen and cartilage oligomeric matrix protein (COMP) during cartilage matrix formation. METHODS: Primary chondrocytes from mice deficient in type IX collagen and COMP (double-deficient) were cultured in monolayer or alginate beads. Anchorage of matrix proteins, proteoglycan and collagen content, collagen crosslinks, matrix metalloproteinase activity, and mechanical properties of the matrix were measured. Electron microscopy was used to study the formation of fibrillar structures. RESULTS: In cartilage lacking both type IX collagen and COMP, matrilin 3 showed decreased matrix anchorage. Less matrilin 3 was deposited in the matrix of double-deficient chondrocytes, while larger amounts were secreted into the medium. Proteoglycans were less well retained in the matrix formed in alginate cultures, while collagen deposition was not significantly affected. Electron microscopy revealed similar cartilage collagen fibril diameters in the cultures of double-deficient and wild-type chondrocytes. In contrast, a larger fibril diameter was observed in the matrix of chondrocytes deficient in only type IX collagen. CONCLUSION: Our results show that type IX collagen and COMP are involved in matrix assembly by mediating the anchorage and regulating the distribution of other matrix macromolecules such as proteoglycans and matrilins and have counteracting effects on collagen fibril growth. Loss of type IX collagen and COMP leads to matrix aberrations that may make cartilage more susceptible to degeneration.

Proteoglycan production is required in initial stages of new cartilage matrix formation but inhibits integrative cartilage repair.

The optimal stimulus to repair or regenerate cartilage is not known. We therefore modulated collagen deposition, collagen crosslinking and GAG deposition simultaneously during cartilage matrix production and integrative repair, creating more insight into their role in cartilage repair processes. Insulin-like growth factor 1 (IGF-1; increases proteoglycan and collagen synthesis), beta-aminopropionitrile (BAPN; a reversible inhibitor of collagen crosslinking) and para-nitrophenyl-beta-D-xyloside (PNPX; interferes with proteoglycan production) were used. Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without IGF-1, BAPN or PNPX alone and in all possible combinations, followed by 3 weeks in control medium. DNA content, GAG and collagen deposition and collagen crosslinks were determined. Cartilage constructs were cultured under the same conditions and histologically analysed for integration of two opposing cartilage matrices. In alginate cultures, inhibition of collagen crosslinking with BAPN, in combination with promotion of matrix synthesis using IGF1, was most beneficial for matrix deposition. Addition of PNPX was always detrimental for matrix deposition. For integration of opposing cartilage constructs, the combination of BAPN, IGF1 and temporary prevention of proteoglycan formation with PNPX was most beneficial. When a new matrix is produced, proteoglycans are important to retain collagen in the matrix. When two already formed cartilage matrices have to integrate, a temporary absence of proteoglycans and temporary inhibition of collagen crosslinking might be more beneficial in combination with stimulation of collagen production, e.g. by IGF1. Therefore, the choice of soluble factors to promote cartilage regeneration depends on the type of therapy that will be used.

Value of serum cartilage oligomeric matrix protein as a prognostic marker of large-joint damage in rheumatoid arthritis--data from the RAPIT study.

OBJECTIVE: To investigate the utility of serum COMP level measurements as a predictor of future damage of the weight-bearing (large) joints in RA patients participating in intensive exercise. METHODS: Data of the 281 completers of a 2-yr randomized controlled trial (Rheumatoid Arthritis Patients In Training; RAPIT) comparing the effects of usual care physical therapy with high-intensity weight-bearing exercises were analysed. The primary outcome variable was defined as the change in radiological joint damage (Larsen score) of the large joints. Potential predictors of outcome were defined: baseline and change in serum level of COMP after 3 months, baseline radiological damage of the large and small joints, number of months on glucocorticoids, change in disease activity and in physical capacity (aerobic fitness and muscle strength) after 2 yrs, and participation in the exercise group. RESULTS: In cross-sectional evaluation of baseline data, we found strong association between the high serum COMP level and current damage of the large joints. Serum COMP level at baseline, however, was not associated with an increased rate of radiological joint damage after 2 yrs of follow-up. Furthermore, neither interaction between baseline COMP level and participation in exercises, nor change in COMP level after 3 months of exercising were associated with future damage of the large joints. CONCLUSION: Neither baseline serum COMP level nor its individual change after 3 months from start of intensive exercise predict longitudinal progression of damage of the large joints in this population.

Degeneration, inflammation, regeneration, and pain/disability in dogs following destabilization or articular cartilage grooving of the stifle joint.

OBJECTIVE: The most used model for joint instability is the canine anterior cruciate ligament transection (ACLT)-model. The ACLT-model can be extended with a medial meniscectomy (MX) (i.e., ACLT-MX-model) to avoid unintentional, and with that variable, meniscal damage. The present study compares the ACLT-MX-model with the more recently introduced Groove-model on longitudinal measurements of osteophyte formation and gait as a surrogate marker of pain and disability, in addition to structural endpoint parameters. METHODS: Degenerative joint damage was induced Labrador dogs according to the ACLT-MX-model (n=7) or Groove-model (n=7). Every 4 weeks radiographs were taken to analyze osteophyte formation. Every 2 weeks gait was recorded using force-plate analysis. Joints were analyzed for features of degeneration 12 weeks after surgery. RESULTS: Both models showed similar osteophyte formation and gait changes for both experimental and contra-lateral control joints, although more pronounced for the ACLT-MX-model. This was supported by the structural endpoint measurements. Cartilage integrity, chondrocyte activity and synovial inflammation revealed similar characteristics of degenerative joint disease in both groups, again more pronounced in the ACLT-MX-model. CONCLUSIONS: The ACLT-MX-model demonstrates characteristics of joint degeneration that are related to moderate to severe osteoarthritis with clear synovial inflammatory activity. The Groove-model is a less painful and a significantly milder model of joint degeneration. The latter model might be more suitable to study subtle changes as a result of intervention than the more robust ACLT-MX-model.

Degenerated and healthy cartilage are equally vulnerable to blood-induced damage.

BACKGROUND: Joint bleeds have a direct adverse effect on joint cartilage, leading to joint deterioration and, ultimately, to disability. OBJECTIVE: To examine the hypothesis that because degenerated cartilage has a limited repair capacity, it is more susceptible than healthy cartilage to blood-induced cartilage damage. METHODS: Healthy, degenerated (preclinical osteoarthritic) and osteoarthritic (clinically defined) human cartilage was exposed to 10% vol/vol whole blood for 2 days, followed by a recovery period of 12 days in the absence of blood. The effect of exposure to blood on cartilage was determined by measuring proteoglycan synthesis rate, release and content, as well as protease (matrix metalloproteinase (MMP)) activity. RESULTS: In general, exposure to blood led to a decrease in proteoglycan synthesis rate, an increase in the release of proteoglycans and in MMP activity, and therefore, ultimately, in a decrease of the proteoglycan content of the tissue. Impaired cartilage was as least as susceptible as healthy cartilage to this blood-induced damage. CONCLUSION: These results demonstrate that degenerated cartilage is not more susceptible than healthy cartilage to blood-induced damage. Even though these are just in vitro findings, it remains of great importance, also, in joints already affected, to prevent joints bleeds, and when they do occur, to treat them adequately.

Early exercise advances the maturation of glycosaminoglycans and collagen in the extracellular matrix of articular cartilage in the horse.

REASON FOR PERFORMING STUDY: Training at a very young age may influence the characteristics of the collagen network of articular cartilage extracellular matrix (ECM) in horses. OBJECTIVES: To investigate whether increasing workload of foals results in significant changes in the biochemical composition of articular cartilage ECM. METHODS: Thoroughbred foals (n = 33) were divided into 2 different exercise groups from age 10 days-18 months. One group (PASTEX; n = 15) was reared at pasture; the other (CONDEX; n = 18) underwent a specific additional training programme that increased workload by 30%. At mean age 18 months, 6 animals from each group were subjected to euthanasia. The proximal articular surface of the proximal phalanx of the right hindlimb was examined for the presence of damage using the cartilage degeneration index (CDI). Samples were taken from 2 sites with known different loading patterns. Slices were analysed for DNA, glycosaminoglycans (GAG), collagen and post translational modifications of collagen (formation of hydroxylysylpyridinoline [HP] and pentosidine crosslinks, and hydroxylysine [Hyl]), and exercise groups and different sites compared. RESULTS: There were no differences in CDI between PASTEX and CONDEX animals, indicating the absence of extra joint damage due to the exercise regimen. There were site-related differences for most biochemical variables, corroborating earlier reports. All biochemical variables showed differences between PASTEX and CONDEX groups at one of the sites, and some at both. GAG and collagen levels were lower in the CONDEX group whereas Hyl, HP crosslinks and pentosidine crosslinks were higher. CONCLUSIONS AND POTENTIAL RELEVANCE: A measurable effect of the conditioning exercise was demonstrated. The margin between too much and too little work when training foals may be narrower than intuitively presumed.

Relaxin stimulates MMP-2 and alpha-smooth muscle actin expression by human periodontal ligament cells.

The main cells in the periodontal ligament (PDL) are the fibroblasts, which play an important role in periodontal remodelling. Matrix metalloproteinases (MMPs) are largely responsible for the degradation of extracellular matrix proteins in the PDL. Previous studies have indicated that MMP production can be stimulated by the hormone relaxin. This hormone facilitates delivery by softening the connective tissues of the reproductive tract, and it prepares the mammary gland for lactation. Periodontal remodelling takes place during orthodontic tooth movement, which might be enhanced by relaxin. Therefore, we investigated the effects of relaxin on gelatinase expression of human PDL cells. Cultures of human PDL cells were incubated with relaxin. Gelatinase (MMP-2 and -9) expression, alpha-smooth muscle actin expression (alpha-SMA), total MMP activity and DNA content were measured. Both proMMP-2 and active MMP-2 was identified in the cultures. There was a clear trend showing a dose-dependent increase of MMP-2 production, which was significant at 250 ng/ml. Total MMP activity was not affected. A stimulation of alpha-SMA expression was found at 50 ng/ml. The results indicate that relaxin activates human PDL cells by the stimulation of MMP-2 and alpha-smooth muscle actin.

Leflunomide and methotrexate reduce levels of activated matrix metalloproteinases in complexes with alpha2 macroglobulin in serum of rheumatoid arthritis patients.

OBJECTIVE: To analyse the effects of leflunomide and methotrexate treatment on matrix metalloproteinase (MMP) activity levels in alpha2 macroglobulin/MMP (alpha2M/MMP) complexes in the systemic circulation of rheumatoid arthritis (RA) patients. METHODS: A total of 102 RA patients from a prospective, double-blind, randomised clinical trial comparing leflunomide and methotrexate were selected; clinical data and blood samples were collected at baseline, at 4 months and at 1 year. Serum MMP activity levels in alpha2M were quantified using low molecular weight fluorogenic substrates, indicating the proportion of activated MMPs that were not inhibited by specific tissue inhibitors of MMP (TIMP). RESULTS: Patients had active disease as shown by high disease activity score (DAS, mean of 6.9 and 7.0 for methotrexate and leflunomide patients respectively), which was reduced over the study period (4.2 and 5.2 respectively, p<0.001). In leflunomide-treated patients a significant reduction of MMP activity levels was observed as early as at the 4 months timepoint persisting thereafter, whereas in methotrexate-treated patients the reduction was seen at 1 year. CONCLUSION: The results show that systemic levels of activated MMPs are reduced in RA patients upon exposure to leflunomide or methotrexate.