Morning stiffness response with delayed-release prednisone after ineffective course of immediate-release prednisone.

OBJECTIVE: To assess morning stiffness in rheumatoid arthritis (RA) patients switched from immediate-release (IR) to delayed-release (DR) prednisone. METHOD: Circadian Administration of Prednisone in Rheumatoid Arthritis-1 (CAPRA-1) is a 12-week, randomized, multicentre, active-controlled study of morning stiffness that consisted of a double-blind phase and a 9-month open-label extension. Patients receiving IR prednisone with no significant improvement after the double-blind study were switched to DR prednisone. Morning stiffness duration and median absolute and relative changes in pain and global assessment were evaluated (3, 6, and 9 months). RESULTS: In patients switched from IR to DR prednisone (n=110), statistically significant reductions in morning stiffness occurred over 3 months and were sustained for 9 months. Absolute reduction of morning stiffness was ~50 min with >40% relative reduction at each visit. Interleukin (IL)-6 levels were reduced by the same amount. Statistically significant and clinically meaningful mean reductions in morning stiffness were maintained at >67 min at each visit along with significant improvements in pain and patient global assessment. There was no evidence of tachyphylaxis seen over the 9-month study. CONCLUSIONS: Patients receiving disease-modifying anti-rheumatic drugs (DMARDs) and IR prednisone who had not had significant reductions in morning stiffness demonstrated statistically significant and clinically meaningful improvements when switched to DR prednisone.

Threshold pixel size for shape determination of microcalcifications in digital mammography: a pilot study.

The effect of pixel size on shape determination in screening digital mammography systems was studied using a shape identification task as the measured outcome. Ten microcalcifications on screen-films were digitised to a range of pixel sizes (2.5-200 microm) and extracted from computed radiography (CR) images (50 microm) acquired under equivalent imaging conditions. Fifteen observers attempted to identify the shape of each microcalcification at each pixel size. The results were collated to provide a fraction of correct responses vs. pixel size curve for each microcalcification. Averaging over all shapes, pixel values >100 microm lead to a significant decrease in shape determination ability (p < 0.01) for digitised screen-film. For CR images, half the shapes were not properly identified. Hence, although 20-100 microm was sufficient for microcalcification shape determination for digitised screen-film images, 50 microm was only borderline sufficient for the CR digital images.

Using simple mathematical functions to simulate pathological structures--input for digital mammography clinical trial.

In this study a set of structures has been simulated to represent a range of clinically relevant breast cancer mammographic lesions including solid tumours and microcalcifications. All structures have been created using simple random-based mathematical functions and have been inserted into a subset of digital mammography images at appropriate contrast levels into various regions of the breast, including dense fibroglandular and adipose tissue. These structures and their appearance in these clinical images were evaluated in terms of how realistic they looked. They will be used as the input to a large-scale clinical trial designed to examine the effect of significant dose reduction in digital mammography by comparing the detectability of such structures in images acquired at full and quarter automatic exposure control (AEC) dose level and in images with simulated noise levels in between.

Can the average glandular dose in routine digital mammography screening be reduced? A pilot study using revised image quality criteria.

There is a need for tools that in a simple way can be used for the evaluation of image quality related to clinical requirements in mammography. The aim of this work was to adjust the present European image quality criteria to be relevant also for digital mammography images, and to use as simple and as few criteria as possible. A pilot evaluation of the new set of criteria was made with mammograms of 28 women from a General Electric Senographe 2000D full-field digital mammography system. One breast was exposed using the standard automatic exposure mode, the other using about half of that absorbed dose. Three experienced radiologists evaluated the images using visual grading analysis technique. The results indicate that the new quality criteria can be used for the evaluation of image quality related to clinical requirements in digital mammography in a simple way. The results also suggest that absorbed doses for the mammography system used may be substantially reduced.

Clinical evaluation of a new set of image quality criteria for mammography.

The European Commission (EC) quality criteria for screen-film mammography are used as a tool to assess image quality. A new set of criteria was developed and initially tested in a previous study. In the present study, these criteria are further evaluated using screen-film mammograms that have been digitised, manipulated to simulate different image quality levels and reprinted on film. Expert radiologists have evaluated these manipulated images using both the original (EC) and the new criteria. A comparison of three different simulated dose levels reveals that the new criteria yield a larger separation of image criteria scores than the old ones. These results indicate that the new set of image quality criteria has a higher discriminative power than the old set and thus seems to be more suitable for evaluation of image quality in mammography.

What Is the Relationship Between Morning Symptoms and Measures of Disease Activity in Patients With Rheumatoid Arthritis?

OBJECTIVE: Little is known about the relationship between morning symptoms of rheumatoid arthritis (RA) and measures of disease activity currently used to assess RA. Information available from the Circadian Administration of Prednisone in Rheumatoid Arthritis (CAPRA-2) study was used to investigate these relationships. METHODS: CAPRA-2 included 350 patients with RA who were symptomatic despite treatment with disease-modifying antirheumatic drugs, randomized 2:1 to additional treatment with a 5-mg daily dose of delayed-release prednisone or placebo. Pearson's correlations were used to evaluate the relationships between change from baseline in symptoms (duration of morning stiffness, severity of morning stiffness, and intensity of pain on waking) and measures of disease activity (the American College of Rheumatology 20% improvement criteria [ACR20], the Disease Activity Score in 28 joints [DAS28], and the Health Assessment Questionnaire disability index). Correlations were defined as weak (<0.3), moderate (0.3-0.7), or strong (>0.7). RESULTS: There was a strong correlation between the severity of morning stiffness and the intensity of morning pain (Pearson's correlation 0.91, P < 0.001). There was a weak correlation between the duration of morning stiffness and measures of disease activity (0.24-0.28), with moderate correlations between the severity of morning stiffness or intensity of pain on waking and DAS28 or ACR20 scores (0.44-0.48). Severity of morning stiffness showed less variability and a greater effect size than did duration of morning stiffness. CONCLUSION: Morning symptoms and measures of disease activity show weak to moderate correlations. Severity of morning stiffness showed less variability and greater effect size than did duration of morning stiffness. These findings suggest that severity is the preferred construct to measure the impact of morning stiffness in patients with RA, information that is not fully captured in the RA core set.

Determination of Lewis FUT3 gene mutations by PCR using sequence-specific primers enables efficient genotyping of clinical samples.

We have developed a polymerase chain reaction method using sequence-specific primers (PCR-SSP) for rapid and correct genotyping of the common Lewis (FUT3) gene mutations 59T>G, 202T>C, 314C>T, 508G>A, and 1067T>A. The PCR-SSP method was validated on 20 healthy blood donors and 16 non-insulin-dependent diabetic patients. All individuals were in parallel genotyped by our established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The FUT3 genotypes, determined with the PCR-SSP method, were in complete accordance with the results of the PCR-RFLP reference method. The PCR-SSP method could also be adapted to assign the presence of a specific mutation to the respective FUT3 alleles. We found the method to be reliable, rapid and cheap with no requirements for restriction enzyme processing.

Determination of drugs by direct injection of plasma into a biocompatible extraction column based on a protein-entrapped hydrophobic phase.

Drugs were determined by direct injection of plasma samples into a biocompatible extraction column. The column is based on particles with a biocompatible external surface and a hydrophobic internal surface. The pores of the particles are small enough to exclude the protein molecules; the drug molecules can penetrate the porous particle and are retained on the hydrophobic internal surface. Biocompatibility of the particles was obtained by reaction of the external surface with the human plasma protein alpha1-acid glycoprotein. The surface within the pores of the particles contains hydrophobic C8 or C18 groups. The biocompatible extraction column was used in a fully automated system for the determination of ibuprofen, naproxen, propranolol, carbamazepine and phenytoin in plasma. No pressure increase was observed during the analysis of several hundred plasma samples. Plasma concentrations of propranolol in the range 4.5-125 ng/ml were determined with a precision (R.S.D.) of 0.75-1.8%. Linear calibration graphs were observed for the five drugs, and correlation coefficients of 1.0000 were obtained for four of the five model compounds.

Optimization of the separation of enantiomers of basic drugs. Retention mechanisms and dynamic modification of the chiral bonding properties on an alpha 1-acid glycoprotein column.

The chromatographic properties of 29 basic drugs were studied by varying the pH and the concentration of inorganic ions in the mobile phase. It was observed that the chromatographic performance of most hydrophobic basic drug compounds could be strongly enhanced by decreasing the pH in the mobile phase from 7 to 4-6. The enantioselectivity increased and a much faster resolution was obtained. The results indicate that ion exchange and ion-pair distribution may be involved in the retention process of cationic drug enantiomers. Increasing the concentration of acetate and phosphate increases the retention of the enantiomers of the drug compounds. The relative contribution of the two retention processes can be affected by the pH and the nature and the concentration of the ions in the mobile phase. Decreasing the pH reduces the influence of the ion-exchange process since the negative charge of the protein is decreased. The enantioselectivity is also greatly affected by increasing salt concentration.

Direct injection of large volumes of plasma/serum on a new biocompatible extraction column for the determination of atenolol, propranolol and ibuprofen. Mechanisms for the improvement of chromatographic performance.

Very large volumes of serum/plasma can be directly injected to a new extraction column based on particles with a biocompatible outer surface and C18 groups within the pores. The biocompatibility has been obtained by attaching the human plasma protein alpha 1-acid glycoprotein to the outer surface of the particles. The pores are small enough to exclude the plasma protein molecules. Atenolol and propranolol were extracted on the extraction column as ion-pair with octanesulfonic acid as the counterion. The same counterion was used in the analytical mobile phase. A strong improvement of the recovery can be obtained using octanesulfonic acid as counterion in the extraction mobile phase. The recovery of atenolol increased from about 53.5% to about 93.4% using octanesulfonic acid as counterion. The chromatographic performance was also strongly affected by chromatography of the basic drugs as ion-pair with octanesulfonic acid. The improvement was due to trapping in a smaller section of the extraction column and enrichment of the drug on top of the analytical column. The enrichment was due to the transfer of the analyte to the analytical column in a zone with high concentration of counterion. Furthermore, the sample zone is compressed during the migration on the analytical column. The compression effect was caused by the counterion zone, migrating in front of the sample zone, giving the analyte higher retention on the front side than on the back side of the sample zone. Displacement of protein bound drug (ibuprofen) by addition of octanoic acid, was tested in order to study the influence on the recovery and the effect on the chromatographic performance. The recovery was improved and the chromatographic performance was greatly improved. The improvement obtained on the separation efficiency of ibuprofen was due to enrichment on top of the analytical column and compression during the migration through the analytical column. The enrichment was caused by a reduction of pH in the sample--octanoic acid zone transferred from the extraction column. The octanoic acid zone migrated in front of the sample zone giving a lower pH in front of the ibuprofen zone than behind. Thus, higher retention occurred in front of than behind the sample zone, which gave rise to compression. The methods developed for atenolol, propranolol and ibuprofen could be used for the determination of serum/plasma concentrations after single doses of the drugs with very high accuracy and precision. Linear calibration graphs were obtained and the r values were > or = 0.9999.

The influence of dialysis fluid composition on the blood pressure response during dialysis.

To elucidate the relative role of osmolar (sodium) and acetate shifts during dialysis, 6 patients with problems of overhydration underwent rapid ultrafiltration for 1 hr (mean weight reduction 2.0 kg), using the 1 m2 RP 6 dialyzer. Ultrafiltration was carried out at the beginning of each of 5 dialysis treatments at weekly intervals. Ultrafiltration was undertaken without dialysis (controls) and with simultaneous dialysis using acetate (40 mmoles/1) or bicarbonate (25 mmoles/1) in the dialysis fluid with dialyzate sodium concentration of 133 and 145 mmoles/1. The systolic blood pressure and mean arterial pressure which were stable with ultrafiltration only fell slightly when a high dialyzate sodium concentration was used and much further when the dialyzate sodium concentration was kept low. These changes were apparently related to the changes in plasma osmolality. Acetate had no effect on blood pressure at the higher sodium concentration, but a slight (insignificant) additive effect when used in the low-sodium dialyzate. Shifts in osmolality (sodium concentration) seem to be more important than the effect of acetate in inducing dialysis-associated hypotension.

Precipitation of heavy metals from landfill leachates by microbially-produced sulphide.

Four leachates from two landfills in Sweden were treated for the removal of heavy metals with the aid of sulphate-reducing bacteria (SRB). Both continuous and batch experiments were performed. A packed-bed process was used for sulphide production. The metals studied were As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn. The continuous experiments showed that Cd and Cu were most efficiently removed and that Cr was the most difficult to precipitate. In a continuous experiment with one of the leachates, the removal of Cd, Cu and Zn depended upon the retention time in the system. In the batch experiments, precipitation of the metals was a relatively fast process. No significant differences in metal concentrations were found between experiments terminated after a day and those terminated after a week. In a batch experiment involving one of the leachates, the precipitation of Cd and Cu was shown to be dependent upon the metal:sulphide ratio. Removal of the metals increased with an increase in the sulphide:metal ratio up to 45:1. The process with SRB showed an interesting potential for removal of heavy metals from leachates. One of the two leachates for which the highest metal removals were obtained came from a landfill for hazardous waste.

Inhibition of endocytosis blocks Wnt signalling to beta-catenin by promoting dishevelled degradation.

AIM: The Wnt/Frizzled signalling pathway is highly conserved through evolution. Frizzled, the receptors for Wnts, have the topology of seven transmembrane spanning domain receptors. An important means of regulation of these receptors is internalization and desensitization through clathrin-mediated endocytosis. Therefore, we investigated the effects of endocytosis inhibition on Frizzled4-green fluorescent protein (FZD(4)-GFP) localization, dishevelled levels and Wnt-3a signalling to beta-catenin. METHODS: Experiments were performed in the mouse neuronal cell line SN4741 that has previously proven to be valuable for the investigation of Wnt/Frizzled signalling. FZD(4)-GFP distribution has been examined using confocal laser scanning microscopy. Dishevelled protein expression levels and the activation of beta-catenin upon treatment with endocytosis inhibitors (hyperosmolaric sucrose and K(+) depletion), kinase inhibitors and Wnt-3a were analysed by immunoblotting. RESULTS: Hyperosmotic sucrose and K(+) depletion increased the membrane localization of FZD(4)-GFP, and in parallel triggered fast (1-2 h) and almost complete (approx. 95%) degradation of endogenous dishevelled, which was independent of Wnt-induced, CK1-mediated phosphorylation of dishevelled. In addition, dishevelled depletion induced by endocytosis inhibition completely prevented canonical signalling by Wnt-3a to beta-catenin even when osmotic conditions and endocytosis were reverted to normal. CONCLUSIONS: The data provide evidence for a molecular mechanism that could be a basis for a novel negative feedback loop within the Wnt/Frizzled pathway depending on dishevelled degradation. The identification of molecular details of regulatory mechanisms for the Wnt/Frizzled signalling pathway increases our understanding of pathway regulation, which might be of special physiological significance for embryonic development, cancer and neurological disorders.

Global changes in cellular gene expression during bacteriophage PRD1 infection.

Virus-induced changes in cellular gene expression and host physiology have been studied extensively. Still, there are only a few analyses covering the entire viral replication cycle and whole-host gene pool expression at the resolution of a single gene. Here we report changes in Escherichia coli gene expression during bacteriophage PRD1 infection using microarray technology. Relative mRNA levels were systematically measured for over 99% of the host open reading frames throughout the infection cycle. Although drastic modifications could be detected in the expression of individual genes, global changes at the whole-genome level were moderate. Notably, the majority of virus-induced changes took place only after the synthesis of virion components, indicating that there is no major reprogramming of the host during early infection. The most highly induced genes encoded chaparones and other stress-inducible proteins.

Complete genome sequence of the broad host range single-stranded RNA phage PRR1 places it in the Levivirus genus with characteristics shared with Alloleviviruses.

Single-stranded RNA (ssRNA) bacteriophages of the family Leviviridae infect gram-negative bacteria. They are restricted to a single host genus. Phage PRR1 is an exception, having a broad host range due to the promiscuity of the receptor encoded by the IncP plasmid. Here we report the complete genome sequence of PRR1. Three proteins homologous with those of other ssRNA phages, i.e., maturation, coat, and replicase proteins, were identified. A fourth protein has a lysis function. Comparison of PRR1 with other members of the Leviviridae family places PRR1 in the genus Levivirus with some characteristics more similar to those of members of the genus Allolevivirus.

Identification of nine alternatively spliced alpha2,3-sialyltransferase, ST3Gal IV, transcripts and analysis of their expression by RT-PCR and laser-induced fluorescent capillary electrophoresis (LIF-CE) in twenty-one human tissues.

In order to characterise the candidate alpha2,3-sialyltransferases necessary for biosynthesis of the selectin ligand SLe(x) and related antigens we have cloned and sequenced, from peripheral blood leukocytes of single individuals, various transcripts from the human ST3Gal III, IV and VI genes. Our clones have revealed a considerable heterogeneity in transcript isoforms. Among our ST3Gal IV clones we isolated nine alternatively spliced transcripts covering the coding region of the human ST3Gal IV gene (A1, A1 - 12, A1 + 18, A2, A2 - 12, A2 + 18, B, B - 12 and B + 18). Five of these isotranscripts A1 - 12, A1 + 18, A2 - 12, A2 + 18 and B + 18 have not been described before. In order to investigate if the alternatively spliced isotranscripts were specific for human PBL, we analysed the expression by RT-PCR and laser-induced fluorescent capillary electrophoresis (LIF-CE) in twenty other human tissues. We found a tissue specific expression of ST3Gal IV A1, A1 - 12, A1 + 18, A2, A2 - 12, A2 + 18 and B + 18 as well as a general expression of ST3Gal IV B and B - 12 isotranscripts in all tissues examined.

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane.

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.

The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption.

Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry.

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.