Lifetime body mass index, other anthropometric measures of obesity and risk of knee or hip osteoarthritis in the GOAL case-control study.

OBJECTIVE: To examine the risk of large joint osteoarthritis (OA) in those becoming overweight during early adult life, and to assess the risks associated with high body mass index (BMI) and other anthropometric measures of obesity. METHODS: BMI, waist and hip circumference were measured in the GOAL case-control study comprising hip OA cases (n=1007), knee OA cases (n=1042) and asymptomatic controls (n=1121). Retrospective estimates of lifetime weight, body shape and other risk factors were collected using an interview-lead questionnaire. Odds ratios (ORs), adjusted OR (aOR), 95% confidence intervals (CIs) and P values were calculated using logistic regression analysis. RESULTS: BMI was associated with knee OA (aOR 2.68, 95% CI 2.33-3.09, P-trend<0.001) and hip OA (aOR 1.65, 95% CI 1.46-1.87, P-trend<0.001). Those who became overweight earlier in adulthood showed higher risks of lower limb OA (P-trend<0.001 for knee OA and hip OA). Self-reported body shape was also associated with knee OA and hip OA, following a similar pattern to current and life-course BMI measures. Waist:hip ratio (WHR) at time of examination did not associate with OA independently of BMI, except in women-only analysis. Waist circumference was associated with lower limb OA risk. CONCLUSIONS: Becoming overweight earlier in adult life increased the risks of knee OA and hip OA. Different distribution patterns of adiposity may be related to OA risk in women.

Large-scale meta-analysis of interleukin-1 beta and interleukin-1 receptor antagonist polymorphisms on risk of radiographic hip and knee osteoarthritis and severity of knee osteoarthritis.

OBJECTIVE: To clarify the role of common genetic variation in the Interleukin-1ß (IL1B) and Interleukin-1R antagonist (IL1RN) genes on risk of knee and hip osteoarthritis (OA) and severity of knee OA by means of large-scale meta-analyses. METHODS: We searched PubMed for articles assessing the role of IL1B and IL1RN polymorphisms/haplotypes on the risk of hip and/or knee OA. Novel data were included from eight unpublished studies. Meta-analyses were performed using fixed- and random-effects models with a total of 3595 hip OA and 5013 knee OA cases, and 6559 and 9132 controls respectively. The role of ILRN haplotypes on radiographic severity of knee OA was tested in 1918 cases with Kellgren-Lawrence (K/L) 1 or 2 compared to 199 cases with K/L 3 or 4. RESULTS: The meta-analysis of six published studies retrieved from the literature search and eight unpublished studies showed no evidence of association between common genetic variation in the IL1B or IL1RN genes and risk of hip OA or knee OA (P>0.05 for rs16944, rs1143634, rs419598 and haplotype C-G-C (rs1143634, rs16944 and rs419598) previously implicated in risk of hip OA). The C-T-A haplotype formed by rs419598, rs315952 and rs9005, previously implicated in radiographic severity of knee OA, was associated with reduced severity of knee OA (odds ratio (OR)=0.71 95%CI 0.56-0.91; P=0.006, I(2)=74%), and achieved borderline statistical significance in a random-effects model (OR=0.61 95%CI 0.35-1.06 P=0.08). CONCLUSION: Common genetic variation in the Interleukin-1 region is not associated with prevalence of hip or knee OA but our data suggest that IL1RN might have a role in severity of knee OA.

Effects of a metalloproteinase inhibitor on osteochondral angiogenesis, chondropathy and pain behavior in a rat model of osteoarthritis.

OBJECTIVE: To investigate the effects of a matrix metalloproteinase (MMP) inhibitor on joint pathology and pain behavior in the rat meniscal transection (MNX) model of osteoarthritis (OA) and evaluate which aspects of structural disease modification contribute to symptom improvement. METHODS: OA pathology was induced in male Lewis rats, by transecting the medial collateral ligament with (MNX) or without (SHAM) a full thickness cut through the meniscus. MNX animals were orally administered an equipotent MMP 2, 8, 9, 12, 13 inhibitor (0.25, 1 and 5 mg/kg/day) or vehicle from day 1. Chondropathy, osteophytosis, osteochondral vascularity were assessed from toluidine blue stained coronal sections of the total knee joint and weight-bearing asymmetry by incapacitance. Group differences were evaluated using 1-way analysis of variance (ANOVA) and associations as Spearman's correlation coefficients. RESULTS: Treatment with the MMP inhibitor reduced weight-bearing asymmetry from day 14 onwards, and attenuated chondropathy (both P<0.05). Osteochondral vascularity was elevated in MNX compared with SHAM-operated animals (P<0.001) and reduced, dose-dependently, by MMP inhibitor treatment (r=-0.89, P<0.05). Reduced osteochondral vascularity and chondropathy were associated with the amelioration of weight-bearing asymmetry (both P<0.05). CONCLUSION: Here we show that treatment with a MMP inhibitor reduces joint damage, osteochondral angiogenesis and behavioral evidence of pain. The association between osteochondral angiogenesis and pain behavior may be explained by perivascular nerve growth or stimulation of subchondral nerves following loss of osteochondral integrity. Our data suggest that targeting angiogenesis may have utility in the treatment of pain associated with structural damage in OA.

Beta2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle does not modulate disease severity in the rodent meniscectomy model of osteoarthritis.

OBJECTIVE: To examine whether beta2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle can modulate the severity of osteoarthritis (OA) in the rodent meniscectomy (MNX) model. METHODS: Male Lewis rats were subcutaneously administered with 1.5 mg/kg/day clenbuterol hydrochloride (n=15) or saline vehicle (n=20) for 14 days. Following pre-treatment, five animals from each group were sacrificed to assess the immediate effects of clenbuterol. The remaining animals underwent either invasive knee surgery (clenbuterol pre-treated n=10; saline pre-treated n=10) or a sham control surgical procedure (saline pre-treated n=5). During disease initiation and progression, weight bearing was assessed by hindlimb loading. Myosin heavy chain (MHC) protein isoforms were quantified by silver stained SDS PAGE. OA severity was graded by assessment of toluidine blue stained step coronal sections of the total knee joint. RESULTS: Clenbuterol treatment resulted in an increase in total bodyweight, growth rate and in quadriceps skeletal muscle mass. Meniscal surgery resulted in the development of OA-like lesions, changes to weight bearing, and changes in MHC protein expression in the quadriceps. Clenbuterol-induced skeletal muscle hypertrophy had no effect on either weight bearing or articular pathology following MNX surgery. CONCLUSIONS: Our data reveal that clenbuterol-induced skeletal muscle hypertrophy is unable to mimic the beneficial clinical effects of increased musculature derived through targeted strength training in humans, in a rodent model of MNX-induced OA. In addition we observed fibre-type switching to "slow twitch" in the quadriceps muscle during the induction of OA that warrants further investigation as to its relationship to joint stability.

Can cartilage loss be detected in knee osteoarthritis (OA) patients with 3-6 months' observation using advanced image analysis of 3T MRI?

PURPOSE: Prior investigations of magnetic resonance imaging (MRI) biomarkers of cartilage loss in knee osteoarthritis (OA) suggest that trials of interventions which affect this biomarker with adequate statistical power would require large clinical studies of 1-2 years duration. We hypothesized that smaller, shorter duration, "Proof of Concept" (PoC) studies might be achievable by: (1) selecting a population at high risk of rapid medial tibio-femoral (TF) progression, in conjunction with; (2) high-field MRI (3T), and; (3) using advanced image analysis. The primary outcome was the cartilage thickness in the central medial femur. METHODS: Multi-centre, non-randomized, observational cohort study at four sites in the US. Eligible participants were females with knee pain, a body mass index (BMI)> or =25 kg/m(2), symptomatic radiographic evidence of medial TF OA, and varus mal-alignment. The 29 participants had a mean age of 62 years, mean BMI of 36 kg/m(2), with eight index knees graded as Kellgren-Lawrence (K&L)=2 and 21 as K&L=3. Eligible participants had four MRI scans of one knee: two MRIs (1 week apart) were acquired as a baseline with follow-up MRI at 3 and 6 months. A trained operator, blind to time-point but not subject, manually segmented the cartilage from the Dual Echo Steady State water excitation MR images. Anatomically corresponding regions of interest were identified on each image by using a three-dimensional statistical shape model of the endosteal bone surface, and the cartilage thickness (with areas denuded of cartilage included as having zero thickness - ThCtAB) within each region was calculated. The percentage change from baseline at 3 and 6 months was assessed using a log-scale analysis of variance (ANOVA) model including baseline as a covariate. The primary outcome was the change in cartilage thickness within the aspect of central medial femoral condyle exposed within the meniscal window (w) during articulation, neglecting cartilage edges [nuclear (n)] (nwcMF x ThCtAB), with changes in other regions considered as secondary endpoints. RESULTS: Anatomical mal-alignment ranged from -1.9 degrees to 6.3 degrees , with mean 0.9 degrees . With one exception, no changes in ThCtAB were detected at the 5% level for any of the regions of interest on the TF joint at 3 or 6 months of follow-up. The change in the primary variable (nwcMF x ThCtAB) from (mean) baseline at 3 months from the log-scale ANOVA model was -2.1% [95% confidence interval (CI) (-4.4%, +0.2%)]. The change over 6 months was 0.0% [95% CI (-2.7%, +2.8%)]. The 95% CI for the change from baseline did not include zero for the cartilage thickness within the meniscal window of the lateral tibia (wLT x ThCtAB) at 6 month follow-up (-1.5%, 95% CI [-2.9, -0.2]), but was not significant at the 5% level after correction for multiple comparisons. CONCLUSIONS: The small inconsistent compartment changes, and the relatively high variabilities in cartilage thickness changes seen over time in this study, provide no additional confidence for a 3- or 6-month PoC study using a patient population selected on the basis of risk for rapid progression with the MRI acquisition and analyses employed.

A meta-analysis of interleukin-6 promoter polymorphisms on risk of hip and knee osteoarthritis.

OBJECTIVE: Interleukin-6 is a pro-inflammatory cytokine involved in the pathogenesis of osteoarthritis (OA). We investigated the role of two single nucleotide polymorphisms (SNPs) mapping to the promoter of the IL-6 gene on genetic susceptibility to hip and knee OA. METHODS: The -174G/C (rs1800795) and -597G/A (rs1800797) SNPs, implicated in the literature in risk of hip and hand OA, were genotyped in 2511 controls, 1101 hip OA cases and 1904 knee OA cases from four cohorts from the UK and Estonia. Data were analysed in conjuntion with published data on rs1800797 from the Genetics of OA and Lifestyle study (UK) on 791 controls, 1034 knee and 997 hip OA cases and rs1800795 data on 75 hip OA cases and 96 controls from Italy. Cases included both radiographic OA only and radiographic and symptomatic OA. Fixed and random-effects meta-analysis models were tested. RESULTS: No significant association was found with hip OA or knee OA with either SNP nor with the haplotypes formed by them. For individual SNPs the smallest P-value for hip OA was observed using a random-effects model for rs1800795 OR(Gallele)=1.066 (95% CI 0.89-1.28) P<0.49, and significant heterogeneity between cohorts (I(2)=65%, P<0.034) was detected. For knee OA the smallest P-value was seen for rs1800797 OR(Aallele)=1.055 (95%CI 0.98-1.12) P<0.18, no significant heterogeneity was observed (I(2)=0%, P<0.68). CONCLUSIONS: Our data do not support a role for the -174 and -597 IL-6 promoter polymorphisms in genetic susceptibility to knee or hip OA in Caucasian populations.

Attempt to replicate published genetic associations in a large, well-defined osteoarthritis case-control population (the GOAL study).

OBJECTIVE: Published studies have tested over 90 genes for association with osteoarthritis (OA), but few positives reported have been independently replicated. Using a new case-control study, our aim was to attempt the replication of findings from 12 genes reported to have significant genetic association with OA and to further examine the role of genetic variation in six of these genes. METHODS: A case-control study was undertaken in Nottingham, UK. Hospital-referred index cases with symptomatic, radiographic OA (ROA) of the knee (n=1040) or hip (n=1004) were recruited. Asymptomatic controls (n=1123) were recruited from intravenous urography waiting lists and screened for radiographic hip and knee OA. Sixty-eight polymorphisms were genotyped in IL1A, IL1B, IL1RN, IL4R, IL6, COL2A1, ADAM12, ASPN, IGF1, TGFB1, ESR1 and VDR. Statistical analysis compared allele or genotype frequencies of these polymorphisms in all asymptomatic controls and the subset of controls without ROA vs all OA, knee OA and hip OA. The analyses were adjusted for age, gender and body mass index. RESULTS: We were unable to replicate any of the published genetic associations investigated. Our extended exploratory analyses identified some associations between polymorphisms in TGFB1, IGF1 and IL1RN and OA; but the strength of evidence varied with the control group used. CONCLUSION: Lack of replication is common and could be due to differences in study design, phenotype, populations examined or the occurrence of false positives in the initial study. Variants within TGFB1, IGF1 and IL1RN could have a role in OA susceptibility; however, replication of these findings is required in an independent study.

Radiographic assessment of the index to ring finger ratio (2D:4D) in adults.

The smaller index to ring finger (2D:4D) ratio has been considered as a 'male finger pattern' and is associated with sporting ability and a number of conditions. However, the ratio may vary according to what is measured, the hand selected and the method used. This study aimed to determine: (1) which bones (phalanges, metacarpals or both) account for variation in the 2D:4D ratio; (2) whether the ratio shows right-left symmetry or relates to hand dominance; and (3) the correlation between visual classification and measured determinations of the ratio based on radiographs. Hand radiographs obtained as part of a large osteoarthritis genetic study were examined. Each hand was classified visually into three types according to the relative length of the index and ring finger: Type 1 (index longer than ring), Type 2 (index = ring) and Type 3 (index shorter than ring). For both index and ring fingers we measured (1) from base of proximal to tip of distal phalanx and (2) metacarpal length. Reproducibility of the classification and measurements were examined using kappa and intraclass correlation coefficient; symmetry between left and right hands was examined using Bland and Altman's agreement analysis; and correlation between visual classification and 2D:4D ratio data was analysed using the anova linearity test. Data were obtained from 3172 radiographs (1636 men, 1536 women; mean age 67 +/- 7.9 years, range 45-86 years). Prevalence of Type 3 hand was 61% in men and 37% in women (P < 0.001). Men had smaller 2D:4D ratios than women for phalanges (0.908 versus 0.922, P < 0.01), metacarpals (1.152 versus 1.157, P < 0.01) and the sum of phalanges plus metacarpals (1.005 versus 1.015, P < 0.01). The mean difference between right and left was -0.001 (95% limit of agreement -0.035, 0.032) for the phalangeal ratio and 0.003 (95% limit of agreement -0.051 to 0.057) for the metacarpal ratio. The 2D:4D ratio did not associate with handedness or age. There was a linear trend between the visual classification of hand type and the 2D:4D ratio data (P < 0.001). More technical difficulties (due to positioning, finger trauma, osteoarthritis) were encountered with the phalangeal ratio and visual categorization than with the metacarpal ratio: the latter could be measured in 98.7% of the study population. We concluded that measured 2D:4D ratios and visual categorization can be derived from hand radiographs. The phalanges and metacarpals both contribute to the variation in 2D:4D ratio with smaller ratios observed in men than in women. The ratio is symmetrical with only very small differences between right and left hands. Visual classification may be a useful simple tool for future epidemiological studies but is more prone to bias from positioning than direct measurement. If radiographs are used for this purpose, we recommend the metacarpal ratio with measurement of a single index hand or an average of both as it is least affected by bias from malpositioning, trauma or common joint disease.

Aggrecan is degraded by matrix metalloproteinases in human arthritis. Evidence that matrix metalloproteinase and aggrecanase activities can be independent.

Proteolytic degradation of aggrecan is a hallmark of the pathology of arthritis, yet the identity of the enzyme(s) in cartilage responsible for this degradation is unknown. Previous studies have suggested that the matrix metalloproteinases (MMPs) may be involved but there has been no definitive evidence for their direct action in the proteolysis of aggrecan in human arthritis. We now show unequivocally that aggrecan fragments derived from the specific action of MMPs can be detected in synovial fluids from patients with both inflammatory and noninflammatory arthritis, with a neoepitope monoclonal antibody AF-28 that detects the NH2-terminal sequence F342FGVG.... The synovial fluid MMP fragments were of low buoyant density and distributed exclusively at the top of cesium chloride density gradients, suggesting that these fragments lacked chondroitin sulfate chains. AF-28 immunoblotting of synovial fluid aggrecan fragments revealed a population of small AF-28 fragments of 30-50 kD. Based on their size relative to characterized products of an MMP-8 digest (Fosang, A.J., K. Last, P. Gardiner, D.C. Jackson, and L. Brown. 1995, Biochem. J. 310:337-343), these AF-28 fragments were derived from proteinase cleavage at, or near, the ...ITEGE373 / ARGSV... aggrecanase site. Immunodetection with polyclonal anti-ITEGE antiserum revealed that these fragments lacked the ...ITEGE374 COOH terminus and were not therefore products of aggrecanase action. The same fluid samples contained a broad 68-90-kD G1 fragment that contained the COOH-terminal ...ITEGE374 neoepitope. The results suggest that in some circumstances, despite extensive proteolysis of the core protein, aggrecan molecules may be cleaved by MMPs or aggrecanase in the interglobular domain, but not both.

Pain in knee osteoarthritis is associated with variation in the neurokinin 1/substance P receptor (TACR1) gene.

BACKGROUND: Substance P (SP) is a pain- and inflammation-related neuropeptide which preferentially binds to the neurokinin receptor 1 (NK1 ). SP and NK1 receptors have been implicated in joint pain, inflammation and damage in animal models and human studies of osteoarthritis (OA). The aim of this study was to test if genetic variation at the neurokinin 1 receptor gene (TACR1) is associated with pain in individuals with radiographic knee OA. METHODS: Participants from the Genetics of OA and Lifestyle study were used for the discovery group (n = 1615). Genotype data for six SNPs selected to cover most variation in the TACR1 gene were used to test for an association with symptomatic OA. Replication analysis was performed using data from the Chingford 1000 Women Study, Hertfordshire Cohort Study, Tasmanian Older Adult Cohort Study and the Clearwater OA Study. In total, n = 1715 symptomatic OA and n = 735 asymptomatic OA individuals were analysed. RESULTS: Out of six SNPs tested in the TACR1 gene, one (rs11688000) showed a nominally significant association with a decreased risk of symptomatic OA in the discovery cohort. This was then replicated in four additional cohorts. After adjusting for age, gender, body mass index and radiographic severity, the G (minor) allele at rs11688000 was associated with a decreased risk of symptomatic OA compared to asymptomatic OA cases (p = 9.90 × 10-4 , OR = 0.79 95% 0.68-0.90 after meta-analysis). CONCLUSIONS: This study supports a contribution from the TACR1 gene in human OA pain, supporting further investigation of this gene's function in OA. SIGNIFICANCE: This study contributes to the knowledge of the genetics of painful osteoarthritis, a condition which affects millions of individuals worldwide. Specifically, a contribution from the TACR1 gene to modulating pain sensitivity in osteoarthritis is suggested.

Does Chondrocalcinosis Associate With a Distinct Radiographic Phenotype of Osteoarthritis in Knees and Hips? A Case-Control Study.

OBJECTIVE: To examine if knee chondrocalcinosis (CC), hip CC, or CC at distant joints associates with a distinct radiographic phenotype of osteoarthritis (OA) in knees or hips. METHODS: We conducted a case-control study using data from the Genetics of Osteoarthritis and Lifestyle (GOAL) study (n = 3,170). All participants of the GOAL study had radiographs of knees, hands, and pelvis, which have been scored for CC and for individual radiographic features of OA. For this study, cases had radiographic OA and CC, and controls had radiographic OA without CC at the index joint. Data for knees and hips were analyzed separately. Binary logistic regression was used to examine the association between each radiographic phenotype and CC in joints with OA. Generalized estimating equation analysis was used to account for correlated data. RESULTS: Knee CC, and CC at any distant joint (without knee CC), associated with attrition in knee OA (adjusted odds ratio 2.32 [95% confidence interval 1.42-3.79] and 2.42 [1.41-4.13], respectively). There was no association between knee CC and osteophytosis or joint space narrowing (JSN) in knees with OA. Hip CC associated negatively with the summated osteophyte score and minimum JSN in hip OA. However, in hips with OA, CC did not associate with cysts or sclerosis. Additionally, distant joint CC did not associate with any structural change in hip OA. CONCLUSION: This study demonstrates that knee CC and CC at distant joints associate with attrition in knee OA, and hip CC associates with a milder hip OA phenotype. There was no evidence that CC associates with a hypertrophic OA phenotype.

The loosely bound non-histone chromosomal proteins of rat prostate in androgen action.

Rat prostatic chromatin was fractionated by sequential salt extractions into 0.35 M NaCl-soluble (the loosely bound non-histone chromosomal proteins), 2 M NaCl-soluble and -insoluble (the residual proteins) fractions. Homologous and heterologous chromatins were reconstituted with these chromosomal fractions from castrated rats injected with testosterone for 2 h and from castrated control. Chromatin reconstituted with all hormone-treated fractions showed 30% more binding with polylysine and transcriptional template activity than did chromatin reconstituted with castrated fractions. Similar analyses of reconstituted heterologous chromatins indicated that the androgen-induced changes in chromatin were mainly determined by the androgenic state of the 0.35 M NaCl-soluble fraction. The 0.35 M NaCl-soluble fraction also contained 18% more total high mobility group protein than did the corresponding castrated fraction. Limited digestion of 5 alpha-[3H]dihydrotestosterone-bound nuclei with pancreatic DNAase 1 to 5% acid-soluble resulted in a rapid release of the nuclear-bound 3H-labelled androgen and protein, in contrast to similar digestion with microsomal nuclease. Since a large portion of the translocation androgen-receptor is bound to protein(s) in the 0.35 M NaCl-soluble fraction, these results suggest that the component(s) in the 0.35 M NaCl-soluble fraction to which androgen-receptor binds is associated with actively transcribing sequences on chromatin.

Measurement of cartilage volumes in rheumatoid arthritis using MRI.

MRI is a valuable imaging modality for assessment of the articular cartilage in rheumatoid arthritis (RA) and is potentially of use in monitoring disease progression and response to therapy. In this study, we investigated the sources of error in volume measurements obtained by segmentation of MR images of knee cartilage in patients with RA and followed cartilage volume in a group of RA patients for 12 months. 23 RA patient volunteers were recruited for knee imaging. Six subjects were imaged at baseline only, six were imaged at baseline and again within an hour in the same imaging session, six subjects were imaged at baseline and 7 days, and 17 subjects were imaged at baseline, 4+/-2 months and 12 months. Imaging was performed at 1.0 T using a three-dimensional spoiled gradient-echo sequence with fat-suppression. Manual image segmentation was performed once or twice on the lateral tibial, medial tibial, patellar and femoral compartment by either one or two segmenters. Coefficients of variation (CoV) for repeated volume measurement of total cartilage were 2.2% (same segmenter, same scan), 5.2% (different segmenter, same scan), 4.9% (same segmenter, different scan, same session), and 4.4% (same segmenter, different scan, different session). Over the 12 month duration of the study there was no significant change in total cartilage volume, nor were there significant changes in volume in any individual compartment. This measurement technique is reproducible, but any net change in cartilage volume over 1 year is very small.

Catalytic activity of ADAM28.

ADAMs are membrane-anchored glycoproteins containing a disintegrin and metalloprotease domain that have important roles in fertilization, development, and diseases such as Alzheimer's dementia. Here we present the first evidence for catalytic activity of ADAM28, a protein that is highly expressed in the epididymis and lymphocytes. Recombinant ADAM28 cleaves myelin basic protein at two sites. The catalytic activity of ADAM28 is not sensitive to tissue inhibitors of metalloproteases 1 and 2, but can be abolished by a mutation in the catalytic site. Catalytically active ADAM28 will be valuable for further studies of its role in sperm maturation and lymphocyte function.

Susceptibility of the cartilage collagens types II, IX and XI to degradation by the cysteine proteinases, cathepsins B and L.

We have investigated the susceptibility of both the helical and non-helical regions of isolated rat chondrosarcoma collagens, types II, IX and XI, to degradation by the cysteine proteinases, cathepsins B and L. Both enzymes degrade these collagens at temperatures from 20 to 37 degrees C and pH values from 3.5 to 7.0. Cleavage occurs only within the non-helical domains unless the helix is destabilized. Cathepsin L is more effective than cathepsin B on a molar basis and they appear to cleave at different sites. Since these cathepsins can degrade cartilage collagens at pH values near neutrality, they may contribute to the destruction of cartilage observed in arthritis.

Monoclonal antibodies to rabbit liver cathepsin B.

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein including the cysteine proteinases, cathepsins H and L.

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates.

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.

Measurement and visualisation of focal cartilage thickness change by MRI in a study of knee osteoarthritis using a novel image analysis tool.

We describe the application of a novel analysis method that provides detailed maps of changes in cartilage thickness measured from MRI scans for individuals and cohorts of patients together with regional measures. A cohort of osteoarthritis patients was imaged using a 1.0 T MR scanner over a 36-month period. Hyaline cartilage was manually segmented from a three-dimensional (3D) spoiled gradient-echo sequence with fat suppression. Representative outlines of the bone surfaces of the distal femur and proximal tibia were automatically generated from T2 weighted images using statistical models of the shape and appearance of the bones. Cartilage thickness was measured from a dense set of points representing the bony surface. The models of the bones provided a common frame of reference, relative to which change maps were generated and aggregated across the cohort and anatomically corresponding subregions of the joint to be identified. In the reproducibility arm involving six patients, the thickness of cartilage had coefficients of variation of 2.66% within the tibiofemoral joint and 2.94% within the medial femoral condyle region. In the 9 patients (6 female, 3 male) who completed the 36-month study, the most striking observation was that lack of change in global measures of cartilage thickness concealed substantial focal changes. Specifically, the cartilage thickness within the tibiofemoral joint decreased by 0.85% per annum (95% CI -2.13% to 0.45%) with the medial femoral condyle as the region with the most significant change, decreasing by 2.43% per annum (uncorrected 95% CI -4.31% to 0.51%).