Non-contact induction heating and SAAP-148 eliminate persisters within MRSA biofilms mimicking a metal implant infection.

Implant-associated infections are the primary cause of complications following orthopaedic surgery. Due to biofilm and persister formation, current treatments, i.e. surgical debridement followed by antibiotics, often fail. There is an urgent need for alternative strategies to combat such infections. Therefore, the present study investigated the effects of non-contact induction heating (NCIH), the antimicrobial peptide SAAP-148 and combinations thereof on bacterial counts in 7 d mature biofilms and in persister-enriched biofilms of methicillin-resistant Staphylococcus aureus (MRSA) on titanium-aluminium-niobium (TAN) discs. Enrichment of persisters was achieved by daily exposure of mature biofilms to high doses of rifampicin and ciprofloxacin for 3 consecutive days. To heat up the TAN discs, a miniaturised induction heater was built and successfully validated. Using this apparatus, NCIH resulting in surface temperatures up to 85 °C eradicated all the bacteria in immature biofilms but not in mature biofilms, whereas persisters were already eliminated at surface temperatures ≥ 70 °C. SAAP-148 at concentrations > 25.6 µmol/L reduced the persister counts in antibiotics-exposed, mature biofilms. As surface temperatures > 60 °C can have detrimental effects on the surrounding tissues, the maximum temperature of NCIH used in combination with SAAP-148 on persisters was set to 60 °C. Results revealed that this combination was slightly more effective than the peptide or NCIH alone in eliminating biofilm-embedded persisters. NCIH and SAAP-148 can be applied both invasively and non-invasively in various treatment scenarios. Together, combinations of NCIH and SAAP-148 might be a promising treatment strategy to combat metal-implant-associated infections.

Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

Pitfalls in the detection of citrullination and carbamylation.

Carbamylation and citrullination are both post-translational modifications against which (auto)antibodies can be detected in sera of rheumatoid arthritis (RA) patients. Carbamylation is the chemical modification of a lysine into a homocitrulline, whereas citrullination is an enzymatic conversion of an arginine into a citrulline. It is difficult to distinguish between the two resulting amino acids due to similarities in structure. However, differentiation between citrulline and homocitrulline is important to understand the antigens that induce antibody production and to determine which modified antigens are present in target tissues. We have observed in literature that conclusions are frequently drawn regarding the citrullination or carbamylation of proteins based on reagents that are not able to distinguish between these two modifications. Therefore, we have analyzed a wide spectrum of methods and describe here which method we consider most optimal to distinguish between citrulline and homocitrulline. We have produced several carbamylated and citrullinated proteins and investigated the specificity of (commercial) antibodies by both ELISA and western blot. Furthermore, detection methods based on chemical modifications, such as the anti-modified citrulline-"Senshu" method and also mass spectrometry were investigated for their capacity to distinguish between carbamylation and citrullination. We observed that some antibodies are able to distinguish between carbamylation and citrullination, but an overlap in reactivity is often present in the commercially available anti-citrulline antibodies. Finally, we conclude that the use of mass spectrometry is currently essential to differentiate between citrullinated and carbamylated proteins present in complex biological samples.

The association between anti-carbamylated protein (anti-CarP) antibodies and radiographic progression in early rheumatoid arthritis: a study exploring replication and the added value to ACPA and rheumatoid factor.

OBJECTIVE: Anti-carbamylated protein (anti-CarP) antibodies are reported to associate with more radiographic progression within the total rheumatoid arthritis (RA) population and anti-citrullinated peptide antibody (ACPA)-negative subgroup. We explored the association of anti-CarP with radiographic progression in RA and aimed to replicate the association and evaluate the added value of anti-CarP antibodies in relation to ACPA and rheumatoid factor (RF). METHODS: 576 Swedish and 628 Dutch patients with RA (2394 and 3247 sets of radiographs, respectively) were longitudinally studied. Replication was restricted to the Swedish patients. In both cohorts, the association of anti-CarP with radiographic progression was determined in strata of patients with similar ACPA and RF status; results of both cohorts were combined in fixed-effect meta-analyses. The net percentage of patients for whom the radiographic progression in 5 years was additionally correctly classified when adding anti-CarP to a model including ACPA and RF was evaluated. RESULTS: Anti-CarP associated with radiographic progression in the total Swedish RA population (beta=1.11 per year, p=8.75×10-13) and in the ACPA-negative subgroup (beta=1.14 per year, p=0.034). Anti-CarP associated with more radiographic progression in the strata of ACPA-positive/RF-negative, ACPA-negative/RF-positive and ACPA-positive/RF-positive patients with RA (respective p values 0.014, 0.019 and 0.0056). A model including ACPA and RF correctly classified 54% and 57% of the patients; adding anti-CarP to this model did not increase these percentages (54% and 56% were correctly classified). CONCLUSIONS: Anti-CarP antibodies associated with more severe radiographic progression in the total and ACPA-negative RA population. Anti-CarP-positivity had a statistically significant additive value to ACPA and RF, but did not improve correct classification of patients.

Anticitrullinated protein antibodies and rheumatoid factor are associated with increased mortality but with different causes of death in patients with rheumatoid arthritis: a longitudinal study in three European cohorts.

OBJECTIVE: Patients with rheumatoid arthritis (RA)-related autoantibodies have an increased mortality rate. Different autoantibodies are frequently co-occurring and it is unclear which autoantibodies associate with increased mortality. In addition, association with different causes of death is thus far unexplored. Both questions were addressed in three early RA populations. METHODS: 2331 patients with early RA included in Better Anti-Rheumatic Farmaco-Therapy cohort (BARFOT) (n=805), Norfolk Arthritis Register (NOAR) (n=678) and Leiden Early Arthritis Clinic cohort (EAC) (n=848) were studied. The presence of anticitrullinated protein antibodies (ACPA), rheumatoid factor (RF) and anticarbamylated protein (anti-CarP) antibodies was studied in relation to all-cause and cause-specific mortality, obtained from national death registers. Cox proportional hazards regression models (adjusted for age, sex, smoking and inclusion year) were constructed per cohort; data were combined in inverse-weighted meta-analyses. RESULTS: During 26 300 person-years of observation, 29% of BARFOT patients, 30% of NOAR and 18% of EAC patients died, corresponding to mortality rates of 24.9, 21.0 and 20.8 per 1000 person-years. The HR for all-cause mortality (95% CI) was 1.48 (1.22 to 1.79) for ACPA, 1.47 (1.22 to 1.78) for RF and 1.33 (1.11 to 1.60) for anti-CarP. When including all three antibodies in one model, RF was associated with all-cause mortality independent of other autoantibodies, HR 1.30 (1.04 to 1.63). When subsequently stratifying for death cause, ACPA positivity associated with increased cardiovascular death, HR 1.52 (1.04 to 2.21), and RF with increased neoplasm-related death, HR 1.64 (1.02 to 2.62), and respiratory disease-related death, HR 1.71 (1.01 to 2.88). CONCLUSIONS: The presence of RF in patients with RA associates with an increased overall mortality rate. Cause-specific mortality rates differed between autoantibodies: ACPA associates with increased cardiovascular death and RF with death related to neoplasm and respiratory disease.

Biomarkers for rheumatoid and psoriatic arthritis.

Rheumatic diseases, such as rheumatoid and psoriatic arthritis are systemic inflammatory conditions characterized by a chronic form of arthritis, often leading to irreversible joint damage. Early treatment for patients with rheumatic diseases is required to reduce or prevent joint injury. However, early diagnosis can be difficult and currently it is not possible to predict which individual patient will develop progressive erosive disease or who may benefit from a specific treatment according to their clinical features at presentation. Biomarkers are therefore required to enable earlier diagnosis and predict prognosis in both rheumatoid arthritis and psoriatic arthritis. In this review we will examine the evidence and current status of established and experimental biomarkers in rheumatoid and psoriatic arthritis for three important purposes; disease diagnosis, prognosis and prediction of response to therapy.

Exercise training programs in Dutch cardiac rehabilitation centres.

PURPOSE: To assess methods for determination of exercise intensity, and to investigate practice variation with respect to the contents, volume and intensity of exercise training programs in Dutch cardiac rehabilitation (CR) centres. METHODS: A paper questionnaire was sent to all Dutch CR centres, consisting of 85 questions for patients with an acute coronary syndrome (ACS) or after coronary revascularisation (Group 1) and for patients with chronic heart failure (CHF, Group 2). RESULTS: CR professionals from 45 centres completed the questionnaires (58 %). Symptom-limited exercise testing was used to determine exercise capacity in 76 % and 64 % of the CR centres in group 1 and group 2, respectively; in these centres, a percentage of the maximum heart rate was the most frequently used exercise parameter (65 % and 56 %, respectively). All CR centres applied aerobic training and the majority applied strength training (64 % in group 1 and 92 % in group 2, respectively). There was a considerable variation in training intensity for both aerobic and strength training, as well as in training volume (1-20 h and 1-18 h respectively). CONCLUSION: Among Dutch CR centres, considerable variation exists in methods for determination of exercise intensity. In addition, there is no uniformity in training volume and intensity.

SMN1 gene duplications are associated with sporadic ALS.

OBJECTIVE: To investigate the role of SMN1 and SMN2 copy number variation and point mutations in amyotrophic lateral sclerosis (ALS) pathogenesis in a large population. METHODS: We conducted a genetic association study including 847 patients with ALS and 984 controls. We used multiplexed ligation-dependent probe amplification (MLPA) assays to determine SMN1 and SMN2 copy numbers and examined effects on disease susceptibility and disease course. Furthermore, we sequenced SMN genes to determine if SMN mutations were more prevalent in patients with ALS. A meta-analysis was performed with results from previous studies. RESULTS: SMN1 duplications were associated with ALS susceptibility (odds ratio [OR] 2.07, 95% confidence interval [CI] 1.34-3.20, p = 0.001). A meta-analysis with previous data including 3,469 individuals showed a similar effect: OR 1.85, 95% CI 1.18-2.90, p = 0.008). SMN1 deletions and SMN2 copy number status were not associated with ALS. SMN1 or SMN2 copy number variants had no effect on survival or the age at onset of the disease. We found no enrichment of SMN point mutations in patients with ALS. CONCLUSIONS: Our data provide firm evidence for a role of common SMN1 duplications in ALS, and raise new questions regarding the disease mechanisms involved.

Improving guideline adherence for cardiac rehabilitation in the Netherlands.

BACKGROUND: In 2004, the Netherlands Society of Cardiology released the current guideline on cardiac rehabilitation. Given its complexity and the involvement of various healthcare disciplines, it was supplemented with a clinical algorithm, serving to facilitate its implementation in daily practice. Although the algorithm was shown to be effective for improving guideline adherence, several shortcomings and deficiencies were revealed. Based on these findings, the clinical algorithm has now been updated. This article describes the process and the changes that were made. METHODS: The revision consisted of three phases. First, the reliability of the measurement instruments included in the 2004 Clinical Algorithm was investigated by evaluating between-centre variations of the baseline assessment data. Second, based on the available evidence, a multidisciplinary expert advisory panel selected items needing revision and provided specific recommendations. Third, a guideline development group decided which revisions were finally included, also taking practical considerations into account. RESULTS: A total of nine items were revised: three because of new scientific insights and six because of the need for more objective measurement instruments. In all revised items, subjective assessment methods were replaced by more objective assessment tools (e.g. symptom-limited exercise instead of clinical judgement). In addition, four new key items were added: screening for anxiety/depression, stress, cardiovascular risk profile and alcohol consumption. CONCLUSION: Based on previously determined shortcomings, the Clinical Algorithm for Cardiac Rehabilitation was thoroughly revised mainly by incorporating more objective assessment methods and by adding several new key areas.

Systematic generation of in vivo G protein-coupled receptor mutants in the rat.

G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

Characterization of the serotonin transporter knockout rat: a selective change in the functioning of the serotonergic system.

Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT-/-) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT-/- rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in non-serotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis.

Deficits in the coordination of agonist and antagonist muscles in stroke patients: implications for normal motor control.

Movement impairments about a single joint in stroke patients may be related to deficits in the central regulation of stretch reflex (SR) thresholds of agonist and antagonist muscles. One boundary of the SR threshold range for elbow flexor and extensor muscles was measured in hemiparetic subjects by analysing electromyographic activity during stretching of relaxed muscles at seven different velocities. For each velocity, dynamic SR thresholds were measured as angles at which electromyographic activity appeared. These data were used to determine the sensitivity of the threshold to velocity and the static SR thresholds for flexors and extensors. In contrast to relaxed muscles in healthy subjects, static flexor and extensor thresholds lay within the physiological range in 11/12 and 4/12 subjects, respectively. This implies that, in the range between the static SR threshold and one of the physiological joint limits, relaxation of the muscle was impossible. Subjects then made slow movements against different loads to determine their ranges of active movement. Maximal flexor and extensor torques were lower in hemiparetic subjects throughout the angular range. In some subjects, ranges were found in which no active torque could be produced in either extensor or both muscle groups. These ranges were related to the boundary values of SR thresholds found during passive muscle stretch. The range in which reciprocally organized agonist and antagonist muscle activity could be generated was limited in all but one subject. When attempting to produce torque from positions outside their measured range of movement, excessive muscle coactivation occurred, typically producing no or paradoxical motion in the opposite direction. Results suggest a relationship between spasticity measured at rest and the movement deficit in stroke by demonstrating a link between motor deficits and control deficits in the central regulation of individual SR thresholds.

Association behavior of native beta-lactoglobulin.

The association behavior of beta-lactoglobulin has been studied by small-angle neutron scattering as a function of protein concentration, temperature, pH, and NaCl concentration of the solution. By indirect Fourier transformation of the spectra, pair-distance distribution functions for the various samples were obtained. These functions provided information on the maximum size, the weight-averaged molecular mass, and the z-averaged radius of gyration of the beta-lactoglobulin particles. At room temperature and pH values below 4 and above 5.2 the protein consists predominantly of monomers and dimers, consistent with literature. In these pH regimes the formation of dimers is favored upon increasing ionic strength and decreasing protein charge (pH values closer to the isoelectric point of the protein). Around pH 4.7, larger oligomeric structures are formed, enhanced by a decrease in temperature and a decrease in ionic strength. beta-Lactoglobulin A associates more strongly than beta-lactoglobulin B. Surprisingly, at pH 6.9 larger structures than dimers seem to be formed at high protein concentrations (> 30 mg mL-1).

Aggregation of beta-lactoglobulin and influence of D2O.

The conformational stability of beta-lactoglobulin increases in D2O over that in H2O. This is concluded from an increase in peak temperature by about 3 degrees C of differential scanning calorimetry (DSC) thermograms and from a decrease in overall aggregation rate. However, effects of pH and salt concentration on the heat-induced aggregation (reaction kinetics, DSC thermograms and aggregate growth) are similar in H2O and D2O. This indicates that the mechanism of heat-induced aggregation of beta-lactoglobulin is not significantly affected by replacement of H2O with D2O.

Gelation of proteins from milk.

We give a quantitative treatment of the destabilization of three types of milk protein dispersions. For this we consider the proteins as adhesive-hard-sphere bio-colloids. If the attractive interactions become strong enough the system passes the percolation threshold and gels. Macroscopic properties of these gels are studied by measuring viscoelasticity and permeability coefficients. These coefficients are related to structural (fractal) properties of the gels which were measured using scattering and confocal scanning laser microscopy (CLSM) techniques. The behaviour of the protein gels can be understood on a qualitative level.