Tumour necrosis factor-α/etanercept complexes in serum predict long-term efficacy of etanercept treatment in seronegative rheumatoid arthritis.

OBJECTIVE: To study whether serum levels of tumour necrosis factor-α (TNF-α), free or bound to etanercept, in biological-naïve adults with rheumatoid arthritis (RA) could predict the long-term efficacy of etanercept, measured as drug survival. METHOD: We identified 145 biological-naïve patients with RA starting treatment with etanercept at the Department of Rheumatology, Skåne University Hospital (1999-2008), of whom 16 had seronegative and 129 seropositive RA. TNF-α in serum was quantified using enzyme-linked immunosorbent assay in samples from the onset of treatment and at 6 week follow-up. Drug survival time was used to evaluate the long-term efficacy of etanercept. RESULTS: Levels of TNF-α were significantly increased at follow-up compared to at the start. At the 6 week follow-up, circulating TNF-α mainly comprised TNF-α in complex with etanercept. Longer drug survival time correlated with increased TNF-α at 6 week follow-up in the patients with seronegative RA, but not in the seropositive patients. CONCLUSION: We demonstrated that levels of circulating TNF-α increased in almost all individuals after initiation of treatment with etanercept and that this increase mainly comprised TNF-α in complex with etanercept. More importantly, this increase may predict drug survival in adults with seronegative, but not seropositive, RA and suggests that measuring TNF-α/etanercept complexes in serum may be relevant in patients with seronegative RA.

Diurnal variation in serum levels of cartilage oligomeric matrix protein in patients with knee osteoarthritis or rheumatoid arthritis.

OBJECTIVE: To monitor changes in serum concentrations of cartilage oligomeric matrix protein (COMP) during a 24-h period to determine any diurnal variation, and to estimate the half life of COMP in the circulation in patients with symptomatic knee osteoarthritis and in those with rheumatoid arthritis. METHODS: Serum samples were drawn every 4 h (7 samples/patient over 24 h) in 10 patients with knee osteoarthritis and 14 patients with rheumatoid arthritis. Osteoarthritis was defined radiographically and clinically (American College of Rheumatology (ACR) criteria) and rheumatoid arthritis according to the 1987 ACR criteria. Serum COMP was measured by sandwich ELISA. A statistical model for the diurnal variation in the COMP levels was developed using the computer program NONMEM. RESULTS: No considerable changes in COMP levels were observed during the day between 08:00 and 21:00 in either group. A significant decrease in serum COMP was apparent during bed rest at night, reaching the lowest levels between 04:00 and 05:00 (p<0.03 or better v all other time points) in patients with osteoarthritis and in those with rheumatoid arthritis. From the rate of decreasing serum COMP levels, a putative half life of COMP in the circulation was estimated to be 7.4 h. CONCLUSION: During normal daytime activities, serum COMP levels are constant. The decrease during the night indicates a rapid elimination of COMP once it has reached the circulation. The stable COMP levels during the day suggest that it is not necessary to further standardise the time of serum sampling in clinical practice.

CNI-1493, an inhibitor of proinflammatory cytokines, retards cartilage destruction in rats with collagen induced arthritis.

OBJECTIVE: To investigate if administration of CNI-1493, an inhibitor of the synthesis of proinflammatory cytokines and NO, protects against development of joint destruction in collagen induced arthritis (CIA) in rats. METHODS: In a placebo controlled experiment, CNI-1493 was given once daily intraperitoneally after onset of clinical arthritis in DA rats. Disease progression was studied by clinical scoring of arthritis, serial measurement of serum levels of COMP, and histological examination of joints. RESULTS: Clinical signs of arthritis were significantly reduced in the CNI-1493 treated group of rats in comparison with the placebo treated group. Histological examinations of paws demonstrated a significant reduction of cartilage destruction in the CNI-1493 treated group, but marked destruction of cartilage in the placebo group. Serum levels of COMP increased in the placebo group, whereas in the CNI-1493 treated group levels were low and decreased significantly during the observation time. CONCLUSIONS: Treatment with CNI-1493 provides efficient protection against synovial inflammation and cartilage destruction when used therapeutically in CIA. The protective effect against cartilage destruction can be monitored by measuring serum COMP. These observations make CNI-1493 an attractive candidate for therapeutic studies in human arthritis, and COMP an attractive serum marker for monitoring joint protective effects.

Subgingival plaque microbiota in Saudi Arabians after use of miswak chewing stick and toothbrush.

BACKGROUND: The chewing stick, the miswak, is used in many developing countries as the traditional means for oral hygiene. It is prepared from the roots, twigs and stem of Salvadora persica or other alternative local plants. OBJECTIVES: To compare the effects of the chewing stick miswak (from S. persica) and toothbrush on subgingival plaque microflora among Saudi Arabian individuals. Further, to investigate whether components extracted from S. persica may interfere with the subgingival plaque micro-organisms. MATERIAL AND METHODS: Fifteen healthy Saudi Arabian male volunteers aged 21-36 years were included in a single-blind, randomized cross-over study. The participants were taught how to use each device properly. Plaque sampling for DNA test was performed at the baseline, 1 week after professional tooth cleaning, and after 3 weeks of either miswak or toothbrush use. Identification and quantification of microbial species were performed by the checkerboard method, using whole genomic, digoxigenin-labelled DNA probes. Inhibition zones around miswak were examined on agar plates with Actinobacillus actinomycetemcomitans and the leukotoxicity of this bacterium was analyzed in a bioassay with macrophages+/-extracts of miswak. RESULTS: Miswak and toothbrushing had a similar influence on the levels of the subgingival microbiota. However, A. actinomycetemcomitans was significantly more reduced by miswak (p<0.05) than by toothbrushing. These results were supported by our in vitro results which, indicated that extracts from S. persica might interfere with the growth and leukotoxicity of A. actinomycetemcomitans. CONCLUSIONS: In contrast to toothbrush use, miswak use significantly reduced the amount of A. actinomycetemcomitans in the subgingival plaque.

Corticosteroid treatment of experimental arthritis retards cartilage destruction as determined by histology and serum COMP.

OBJECTIVE: To examine if changes in serum cartilage oligomeric matrix protein (COMP) correlate with the development of cartilage damage, as measured by histological grading, in corticosteroid-treated animals with collagen-induced arthritis (CIA). METHODS: DA rats with established CIA were treated with corticosteroids (betamethasone, 0.1 mg/kg body weight) or placebo (saline) intraperitoneally once daily after reaching an arthritis score exceeding 1. The treatment continued throughout the study. Arthritis progression was monitored by clinical scoring of paws, serial measurements of serum COMP and fibrinogen, and histological grading of paws. RESULTS: Corticosteroid treatment reduced clinical signs of arthritis compared with placebo (arthritis score reduced, P < 0.01 at day 25). Corticosteroid treatment also reduced fibrinogen levels compared with placebo (P < 0.01). The morphological changes in the joint were less severe in the corticosteroid-treated animals (median cartilage score 4 in the placebo group, 0 in the corticosteroid-treated group; P < 0.01). The levels of COMP remained unchanged during treatment in the corticosteroid-treated arthritic animals, whereas an increase in levels of COMP was observed in rats treated with placebo (P < 0.01). There was a correlation between serum COMP and the extent of cartilage destruction at day 25 after immunization (r = 0.77, P < 0.001). CONCLUSIONS: Corticosteroids given therapeutically to arthritic rats diminish joint destruction histologically, and stable serum COMP levels reflect this effect.

Serum concentrations of cartilage oligomeric matrix protein, fibrinogen and hyaluronan distinguish inflammation and cartilage destruction in experimental arthritis in rats.

OBJECTIVES: We investigated if changes in serum/plasma fibrinogen (FIB), hyaluronan (HA) and cartilage oligomeric matrix protein (COMP) levels can be used to differentiate between inflammation and cartilage involvement during arthritis. METHODS: Collagen-induced arthritis (CIA), oil-induced arthritis (OIA) and for comparison, experimental autoimmune encephalitis (EAE) induced in DA rats were investigated. RESULTS: Elevations of FIB concentrations were apparent at days 4-7 post-immunization in both arthritis models reaching a maximum on day 20-21, i.e. before peak arthritis. Elevations of HA in both models were seen shortly before macroscopically apparent arthritis, and peaked at or just before maximal arthritis, i.e. later in CIA than in OIA. COMP levels increased only after onset of arthritis and peaked late in disease (days 34-37), being significantly higher in the more destructive CIA compared with the less destructive OIA. During EAE flares, only FIB levels increased. CONCLUSIONS: FIB is a general inflammation marker, HA appears to be a marker for synovitis and changes in COMP levels appear to reflect the cartilage destruction process.

Light-induced fluorescence studies on dehydration of incipient enamel lesions.

Changes in the hydration state of enamel affect its optical qualities, such as light scattering and fluorescence. In this study, the rate of fluorescence loss was measured when incipient enamel lesions with different de-remineralization history were left to dehydrate. Four groups of lesions were studied. In groups A, B and C, the lesions were prepared in vitro in an acid-gel system. Group A was kept as control, and groups B and C were remineralized (4 weeks) without and with 1 ppm F in solution, respectively. Group D consisted of natural incipient lesions. Enamel fluorescence was measured for all lesions immediately after removal from water and subsequently at short intervals for 30 min. The change in fluorescence with dehydration varied between the groups. In lesions from groups A and B, it followed a double exponential decrease, while in lesions from groups C and D, it followed a mono-exponential decrease. In all groups, the fluorescence of sound surfaces declined mono-exponentially. The 'fractional fluorescence difference', defined as (L(sound) - L(carious) )/L(sound), became constant after periods of dehydration of about 5, 5, 20 and 5 min for groups A to D, respectively. The observation of the change of fluorescence with dehydration should be taken into consideration when planning studies that use fluorescence as an assessment method. However, it might also be used to gain insight into the properties for fluid transport inside the various lesions, relevant to de-remineralization or fluoride treatments.

Quantitative light-induced fluorescence (QLF): a method for assessment of incipient caries lesions.

OBJECTIVES: To review the literature on validation and application of the quantitative light-induced fluorescence (QLF) method for quantitative assessment of early enamel lesions in vivo and in vitro. METHODS: QLF uses light with wavelengths around 405 nm to excite yellow fluorescence at wavelengths above 520 nm. Its diagnostic capacity is based on the mechanism that the intensity of natural fluorescence of a tooth is decreased by scattering due to a caries lesion. The equipment, the data processing and the interaction between equipment and operator are described. RESULTS: The method has been validated by many authors; the results are presented and compared. For artificial lesions, the validation line is curved. For larger mineral losses, the curve is linear with a slope of 10% fluorescence loss corresponding with a mineral loss of 0.15 kg x m(-2). For lesions caused by natural caries, it is tentatively concluded that fluorescence loss is linear to mineral loss with a slope similar to that of artificial lesions. Reliability and reproducibility have been tested in vivo and show interexaminer values of the interclass correlation coefficient, r, of 0.93

Association of chondroadherin with collagen type II.

Chondroadherin is a cell binding, leucine-rich repeat protein found in the territorial matrix of articular cartilage. Several members of the leucine-rich repeat protein family present in the extracellular matrix of e.g. cartilage have been shown to interact with collagen and influence collagen fibrillogenesis. We show that complexes of monomeric collagen type II and chondroadherin can be released under non-denaturing conditions from articular cartilage treated with p-aminophenylmercuric acetate to activate resident matrix metalloproteinases. Purified complexes as well as complexes formed in vitro between recombinant chondroadherin and collagen type II were studied by electron microscopy. Chondroadherin was shown to bind to two sites on collagen type II. The interaction was characterized by surface plasmon resonance analysis showing K(D) values in the nanomolar range. Both chondroadherin and collagen interact with chondrocytes, partly via the same receptor, but give rise to different cellular responses. By also interacting with each other, a complex system is created which may be of functional importance for the communication between the cells and its surrounding matrix and/or in the regulation of collagen fibril assembly.

Chronic pain and difficulty in relaxing postural muscles in patients with fibromyalgia and chronic whiplash associated disorders.

OBJECTIVE: To investigate if muscle tension according to the surface electromyogram (EMG) of the shoulder flexors is increased in consecutive patients with fibromyalgia (FM) or chronic whiplash associated disorders (WAD). METHODS: A total of 59 consecutive patients with FM (n = 36) or chronic WAD (n = 23) performed 100 maximal isokinetic contractions combined with surface electromyography of the trapezius and infraspinatus. A randomized group of pain-free female (n = 27) subjects served as control group. Peak torque initially (Pti) and absolute and relative peak torque at endurance level (PTe, PTer) were registered as output variables, together with the EMG level of unnecessary muscle tension, i.e., the signal amplitude ratio (SAR). RESULTS: The patient groups had a higher level of unnecessary tension initially and at the endurance level. The patients had lower absolute output (PTi and PTe), but the relative levels (PTer) did not differ comparing all 3 groups. Subjects with FM had significantly higher body mass index (BMI) than the other groups. BMI did not influence the SAR but correlated positively with PTi. CONCLUSION: The results confirmed earlier findings that groups of patients with chronic pain have increased muscle tension and decreased output during dynamic activity compared to pain-free controls. However, the results indicated there is heterogeneity within groups of patients with the same chronic pain disorder and that not all patients with chronic pain have increased muscle tension.

Application of quantitative light-induced fluorescence to monitor incipient lesions in caries-active children. A comparative study of remineralisation by fluoride varnish and professional cleaning.

The aim of this study was to apply the quantitative light-induced fluorescence (QLF) method in a randomised controlled study, comparing treatment with fluoride varnish and professional tooth cleaning for remineralisation of white spot lesions in caries-active adolescents. In the fluoride varnish group (n = 13; 32 lesions), professional tooth-cleaning was followed by application of fluoride varnish at the beginning of the study, after 1 wk, and then once every 6 wk for 6 months. The other group (n = 18; 30 lesions) underwent professional tooth-cleaning once every 6 wk for 6 months. Enamel fluorescence was measured at baseline and at each visit. In the fluoride varnish group there was a significant change over time (baseline: 6 months) for both lesion area: and average change in fluorescence (decreased lesion area and increased fluorescence radiance). The corresponding changes in the professional tooth-cleaning group were not significant. There was a significant difference in average change in fluorescence between the two test groups. For lesion area, there was no significant difference, but a tendency towards a difference between the test groups. It was concluded that (a) the QLF method is a sensitive method, suitable for longitudinal quantification of incipient caries lesions on smooth surfaces; and (b) that repeated fluoride applications had a favourable effect on the remineralisation of white spot lesions as measured after 6 months.

How to measure the effects of fluoride treatments in clinical trials? Assessment: modern versus traditional methods.

In recent years there has been a pronounced change in the epidemiology and disease pattern of dental caries. In the current context, traditional methods of caries assessment, discriminating lesions at cavitation, are clinically inappropriate, and obsolete for research requiring detection of a very early phase of mineral loss. Modern prospective caries studies require sensitive methods permitting the measurement of small changes in tooth mineral content, and objective, quantitative measurements of such changes are now possible in a single caries lesion. For longitudinal studies there are noninvasive methods for assessment of new lesions as well as quantitative changes (progression or regression) in existing lesions. Among as yet unresolved issues are improved methods to assess the current activity of a lesion, methods for detection and quantification of secondary caries and root caries, calibration of methodologies between different research institutes, and methods capable of assessment of the whole continuum in the development of a caries lesion, from initial loss of mineral to cavitation.

Validation of DIAGNOdent for quantification of smooth-surface caries: an in vitro study.

The aims were 1) to validate a laser-based device, KaVo DIAGNOdent, for quantification of caries lesions on smooth surfaces, using histopathologic and microradiographic analyses as the gold standard, and 2) to test inter- and intra-observer agreements. We also investigated the influence on DIAGNOdent readings of the storage medium used for extracted teeth. Two observers measured independently the tooth surfaces of 40 extracted premolars that had been stored in thymol-saturated saline. After subsequent storage in neutral-buffered formalin for 14 days they were re-measured. The teeth were then sectioned for histopathologic and microradiographic analysis. The Spearman rank correlation coefficients between lesion depth and DIAGNOdent readings were 0.78-0.83 and 0.85 for teeth stored in thymol-saturated saline and formalin, respectively. Inter- and intra-observer agreements were 0.94 and 0.95 when the teeth were stored in thymol-saturated saline. The DIAGNOdent reading was almost 1.5 times higher for teeth stored in formalin than for those stored in thymol saline. The Spearman rank correlation coefficient between mineral loss in enamel (deltaZ) and DIAGNOdent readings ranged from 0.64 to 0.68. It was concluded that DIAGNOdent may be helpful for assessing smooth-surface caries, but the cut-off points need to be assessed under clinical conditions. The increase in fluorescence associated with storage of teeth in formalin warrants further investigation.

Tuned-aperture computed tomography for detection of occlusal caries.

OBJECTIVES: To evaluate the use of tuned-aperture computed tomography (TACT) for the detection of primary occlusal caries. MATERIALS AND METHODS: The material comprised 76 extracted posterior teeth, 51 with caries. Radiographs were recorded both with the Sens-A-Ray (Regam Medical Systems Int. AB, Sundsvall, Sweden) digital radiographic system and with Kodak EktaSpeed Plus film (Eastman-Kodak Co., Rochester NY, USA) employing an Orthopantomograph OP 100 (Instrumentarium Imaging, Tuusula, Finland) and a Prostyle Intra (Planmeca, Helsinki, Finland) dental unit respectively. The Sens-A-Ray radiographs were used to construct TACT slices and TACT iterative slices. The teeth were subsequently sectioned in 300 microm thick slices. Microradiographs were exposed and analysed with respect to the true presence of lesions. Seven observers subjectively evaluated the radiographs and ROC analyses performed. Conventional radiographs were compared with TACT images by means of the area under the ROC curves, Az. Paired t-test was used to compare Az values. Interobserver agreement was evaluated using the Kendall coefficient and the Friedman Anova. RESULTS: TACT radiographs were significantly better than conventional radiographs for diagnosing all types of occlusal caries combined (P(TACT) (slices)=0.02. P(TACT) (iterative slices)=0.01). However, neither TACT system was significantly better than film for enamel and dentinal caries separately. Observer agreement was moderate. However, observers demonstrated significant systematic differences in their readings (P<0.001). CONCLUSION: TACT may be a feasible method for diagnosing primary occlusal caries.

Release of cartilage and bone macromolecules into synovial fluid: differences between psoriatic arthritis and rheumatoid arthritis.

OBJECTIVE: To elucidate whether differences in the destructive tissue process in cartilage and bone in psoriatic arthritis (PsA) and rheumatoid arthritis (RA) can be recognised by different release patterns of molecular fragments derived from joint tissue. METHODS: Aggrecan, cartilage oligomeric matrix protein (COMP), and bone sialoprotein (BSP) were quantified by immunoassays in knee joint synovial fluid samples. These were obtained early in the disease course of patients with PsA and RA. At the time of arthrocentesis radiographs of their knee and hip joints were normal. RESULTS: At follow up no destruction had developed in the knees and hips of most patients with PsA (n=18), whereas the patients with RA could be separated into one "destructive" group (n=18) and one "non-destructive" group (n=25). Patients with PsA had low synovial fluid aggrecan concentrations (p<0.001 v the RA destructive group) but high COMP concentrations (p<0.01 and p<0.05 v destructive and non-destructive RA groups, respectively). Consequently, the aggrecan/COMP ratio was lowest in the PsA group (p<0.001 and p<0.01 v the destructive and non-destructive RA group, respectively). The synovial fluid concentrations of BSP did not differ between the three patient groups. CONCLUSIONS: The release pattern of aggrecan and COMP, reflecting cartilage turnover, differed between the PsA group and, particularly, the destructive RA group. This suggests that different pathophysiological mechanisms for cartilage involvement operate in these conditions, with different destructive potential. The BSP concentrations did not differ between the patients groups, which indicates similar levels of bone involvement.

Comparison of QLF and DIAGNOdent for quantification of smooth surface caries.

The aim was to compare the detection and quantification of smooth surface caries by KaVo DIAGNOdent and quantitative laser/light-induced fluorescence (QLF) using a laser and a lamp as two different light sources. On 40 premolar teeth extracted from adolescents, 71 non-cavitated approximal surfaces were assessed. For QLF, both mean and maximum fluorescence losses were registered. To provide a gold standard for verification, the teeth were then sectioned and lesion depth, stratified on a five-point scale, was determined by histopathology and microradiography. The correlation between the gold standard and the two methods was assessed by Spearman's rank correlation coefficient. The sensitivity and specificity of the methods for detection of smooth surface caries at D(3) level were also assessed. For lesion depth, correlation with the gold standard was similar for QLF and DIAGNOdent: about 0.85. With respect to dentinal caries detection, sensitivity for DIAGNOdent was 0.75 and specificity 0.96, with a cut-off point of 9. The corresponding values for QLF were 0.94 and 1, with a cut-off value of 20% of fluorescence loss. Spearman's rank correlation coefficients for enamel mineral loss and QLF and DIAGNOdent, respectively, were 0.76 and 0.67. It was concluded that for quantification of smooth surface caries, the methods are of equal merit, but for scientific purposes, QLF offers the advantage of closer correlation with changes in mineral content.

Characterization and validation of diagnostic methods.

Diagnosis is defined as the determination of disease, but not as the determination of the signs and symptoms thereof. The use of modern diagnostic methodology in the clinic is hampered by cost considerations and by the still widespread belief that, e.g. caries lesions and periodontal breakdown are irreversible processes that need to be detected and treated invasively as early as possible, their measurement thus being irrelevant. Modern instrumental and quantitative methods allow early detection and introduction of noninvasive preventive measures to control the development of the disease. Such methods are also very beneficial in clinical research as they may describe the speed of progress or regress of disease. In epidemiology, such methods reduce the classical problem of calibration of observers. Repeatability, reproducibility, accuracy and validity are defined as method-characterizing quantities, for which examples are given. To express the validity of quantitative methods compared with a quantitative gold standard, the use of scatter plots and correlation and regression methods is suggested. Validation of a dichotomous method with a dichotomous gold standard in terms of sensitivity and specificity is discussed. To validate a quantitative method with a dichotomous gold standard, the receiver operating characteristic curve is suggested, with the requirement that the cutoff value should be determined in relation to the use of the method. However, preferably a quantitative method should not be reduced to a dichotomous one by using a cutoff value, but instead all available information should be used by the diagnostician. It is argued that the use of a secondary standard instead of the accepted gold standard usually leads to inadequate results, even when the validity of the secondary standard is known. Finally, it is argued that the choice of a gold standard is a matter of reasoning and weighing of arguments and not of following a prescribed procedure.