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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

RAGE activation induces invasiveness of RA fibroblast-like synoviocytes in vitro.

Ligands for the receptor for advanced glycation endproducts (RAGE) are increased in RA synovial fluid (SF), serum and synovium. Since RAGE is present on fibroblast-like synoviocytes (FLS), the present study investigates whether the RAGE ligands HMGB-1 and AGEs are able to stimulate the characteristic, pathological invasive behaviour of these cells. FLS were obtained during joint replacement surgery. FLS were seeded in serum free medium with HMGB-1 or glycated albumin (BSA-AGE) on transwell filters coated with Matrigel. The lower compartment contained medium with serum as a chemoattractant. After three days, the percentage of invading cells was determined and compared to the control invasion. Stimulation with HMGB-1 increased invasiveness to 125% compared to the control (p = 0.001). Addition of anti-RAGE antibody reduced this back to baseline (98%, p = 0.002). Stimulation with BSA-AGE, another RAGE ligand, increased invasiveness to 150% compared to the control (p = 0.003). Addition of anti RAGE was again able to reduce the increased invasiveness back to baseline (95%, p = 0.008). HMGB-1 and BSA-AGE stimulated the invasiveness of RA-FLS by activation of RAGE. As such, RAGE may be an interesting target for therapy directed at the inhibition of synoviocyte activation.

Fibroblast-like synoviocyte-chondrocyte interaction in cartilage degradation.

OBJECTIVE: In vitro models for joint diseases often focus on a single cell type, such as chondrocytes in osteoarthritis (OA) or fibroblast-like synoviocytes (synoviocytes) in rheumatoid arthritis (RA). However, these joint diseases affect the whole joint and interaction between chondrocytes and synoviocytes may play an important role in disease pathology. The current study was designed to study the use of the alginate recovered chondrocyte method as a model for cartilage degradation and to study interaction between chondrocytes and synoviocytes. METHODS: Bovine chondrocytes were cultured in alginate beads for 1 week, subsequently chondrons were retrieved and seeded into transwells. Every two days cartilage-slices were analysed for proteoglycan content (colorimetric, Blyscan GAG kit), collagen content (HPLC) and collagen HP and LP crosslinking (HPLC). For degradation experiments, monocultures of cartilage-slices labelled with (35)S and cocultures with synoviocytes were stimulated with IL-1beta or TNF-alpha. After 7 days, (35)S release was measured taken as a measure of cartilage degradation. RESULTS: After biochemical analysis, three week old cartilage-like slices were chosen to perform cartilage-degradation experiments. Synoviocytes were able to induce cartilage degradation only in the presence of living chondrocytes. In addition, the cytokines interleukin 1 (IL-1beta) and tumor necrosis factor (TNF-alpha) were only able to induce cartilage degradation by chondrocytes, not by synoviocytes. CONCLUSION: These data indicate that the alginate recovered chondrocyte method provides a novel model for cartilage degradation in which the interaction between synoviocytes and chondrocytes can be studied.

Lysozyme sensitivity of pantothenate-deficient Lactobacillus plantarum grown with exogenous fatty acids.

A pantothenic acid deficiency in Lactobacillus plantarum reduces lipid synthesis, prevents normal uptake and retention of extracellular amino acids and markedly increases sensitivity of these cells to lysozyme induced lysis. Pantothenate-deficient cells provided with exogenous fatty acids synthesize additional lipids and express nearly normal solute transport activities. The present study has shown that such cells retain a heightened sensitivity to lysozyme induced lysis. These observations indicate that the lysozyme sensitivity of pantothenate-deficient cells is not produced as in indirect effect of membrane lipid depletion, but represents an independent consequence of pantothenate insufficiency.

Extracellular matrix changes in early osteochondrotic defects in foals: a key role for collagen?

Osteochondrosis (OC) is the most important developmental orthopaedic disease in the horse. Despite some decades of research, much of the pathogenesis of the disorder remains obscure. Increasing knowledge of articular cartilage development in juvenile animals led to the presumption that the role of collagen in OC might be more important than previously thought. To study collagen characteristics of both cartilage and subchondral bone in young (5 and 11 months of age) horses, samples were taken of subchondral bone and articular cartilage from a group of 43 Dutch Warmblood foals and yearlings that suffered from varying degrees of OC. Based on a histological classification, lesions were graded as early, middle and end stage. Collagen content and some posttranslational modifications (lysyl hydroxylation, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) cross-links) were determined, as was proteoglycan content. Data were compensated for site effects and analysed for differences due to the stage of the lesion. In early lesions total collagen was significantly decreased in both cartilage and subchondral bone of 5- and 11-month-old foals. Also in cartilage, HP cross-linking was reduced in the early lesions of 5- and 11-month-old foals, while LP cross-linking was decreased in subchondral bone of the end-stage lesions of both 5- and 11-month-old foals. Hydroxylysine content was unaffected. Collagen content remained reduced in cartilage from middle- and end-stage lesions, but returned to normal in subchondral bone. In cartilage there was a decrease in proteoglycan content in the end-stage lesions of both age groups. Thus, alterations of the collagen component, but not of the proteoglycan component, of the extracellular matrix might play a role in early OC. More severe lesions show a more general picture of an unspecific repair reaction. Biomarkers of collagen metabolism can be expected to be good candidates for early detection of OC.

Urinary excretion of collagen degradation markers by sows during postpartum uterine involution.

Incomplete uterine involution is the putative cause of the increased embryo mortality and reproductive failure often exhibited by sows that lactate for less than 21 days. Since such short lactation lengths are common in American swine production, an effective technique to monitor the postpartum involution process and test this hypothesis might be valuable. Rapid and extensive catabolism of uterine collagen is essential for normal postpartum involution. The objective of this study was to characterize postpartum excretion of two biochemical markers of collagen degradation. In experiment I, urine samples were collected from five sows every other day from the day before parturition (day -1), through a 21-day lactation, to day 8 postweaning. The collagen crosslinks hydroxylysyl pyridinoline (HP), which is present in many tissues, and lysyl pyridinoline (LP), which is primarily concentrated in bone, were assayed by both ELISA and HPLC. Urinary levels of both free (ELISA) and total (HPLC) HP and LP increased (P < 0.001) approximately two-fold during lactation. The mean molar ratio of total HP:LP increased (P < 0.001) from 6.6 +/- 1.6 at day 1 to a maximum of 10.2 +/- 1.5 at day 7 postpartum and averaged 9.1 +/- 0.3 for the entire sampling period. These data are consistent with a postpartum increase of soft tissue collagen catabolism since bone has a low HP:LP ratio of 4 and soft tissues like the uterus have a high HP:LP ratio of >/=20 because they contain only trace amounts of LP. Since HPLC (total) and ELISA (free) crosslinks estimates were highly correlated (r = 0.85-0.91, P < 0.001) in experiment I, only the less technical ELISA technique was used in experiment II. Urine samples were collected from 21 sows every third day from day 1 to 19 of lactation. Sows from this second group exhibited one of four distinct crosslinks excretion patterns: peak on day 1 (n = 3), peak on day 7 (n = 4), peak on day 10, 13 or 16 (n = 7), or no peak (n = 7). This variation of postpartum crosslinks excretion among sows was not related to parity, body weight, lactation body weight change, litter size, or litter birth weight. Overall, data from experiments I and II indicate that urinary HP does increase postpartum in a pattern temporally consistent with uterine involution. However, significant variation among sows in the magnitude and timing of peak HP excretion was evident.

Study of cartilage and bone layers of the bearing surface of the equine metacarpophalangeal joint relative to different timescales of maturation.

REASONS FOR PERFORMING STUDY: A detailed and comprehensive insight into the normal maturation process of the different tissues that make up functional units of the locomotor system such as joints is necessary to understand the influence of early training on musculoskeletal tissues. OBJECTIVES: To study simultaneously the maturation process in the entire composite structure that makes up the bearing surface of a joint (cartilage, subchondral and trabecular bone) in terms of biochemical changes in the tissues of juvenile horses at 2 differently loaded sites of the metacarpophalangeal joint, compared to a group of mature horses. HYPOTHESIS: In all the structures described above developmental changes may follow a different timescale. METHODS: Age-related changes in biochemical characteristics of the collagen part of the extracellular matrix (hydroxylysine, hydroxyproline, hydroxypyridinum crosslinks) of articular cartilage and of the underlying subchondral and trabecular bone were determined in a group of juvenile horses (n = 13) (Group 1, age 6 months-4 years) and compared to a group of mature horses (n = 30) (Group 2, >4 years). In both bony layers, bone mineral density, ash content and levels of individual minerals were determined. RESULTS: In cartilage, subchondral bone and trabecular bone, virtually all collagen parameters in juvenile horses were already at a similar (stable) level as in mature horses. In both bony layers, bone mineral density, ash- and calcium content were also stable in the mature horses, but continued to increase in the juvenile group. For magnesium there was a decrease in the juvenile animals, followed by a steady state in the mature horses. CONCLUSIONS: In horses age 6 months-4 years, the collagen network of all 3 layers within the joint has already attained a mature biochemical composition, but the mineral composition of both subchondral and trabecular bone continues to develop until approximately age 4 years. POTENTIAL RELEVANCE: The disparity in maturation of the various extracellular matrix components of a joint can be assumed to have consequences for the capacity to sustain load and should hence be taken into account when training or racing young animals.