Evaluation of a new erosion score by musculoskeletal ultrasound in patients with rheumatoid arthritis: is US ready for a new erosion score?

The objectives of this study are to evaluate a new semi-quantitative (0-5) musculoskeletal ultrasound (US) erosion score in patients with rheumatoid arthritis (RA) and to prove its usefulness in the detection of disease activity and success of therapy. Thirty-eight patients with RA (mean disease duration 10.1 ± 11.9 years) were enrolled. Start or change of therapy (DMARD/biologics) was an inclusion criterion. DAS28, laboratory (ESR and CRP) and US data were evaluated before new therapy initiation and after 1, 3, 6 and 12 months. Thirteen joints of the clinically more affected hand and forefoot (wrist and MCP, PIP, MTP joints 2-5) were analyzed for synovitis in grayscale (GS) and power Doppler (PD) US, tenosynovitis/paratenonitis in GS/PDUS (wrist, MCP level) and for erosions. Erosions were analyzed by a new semi-quantitative score (grade 0, no erosion; grade 1, <1 mm, grade 2, 1 to <2 mm; grade 3, 2 to ≤3 mm; grade 4, >3 mm; grade 5, multiple bone erosions). After 12 months, DAS28 decreased from 4.5 to 3.4 (p < 0.001), the synovitis score in GSUS from 26.3 to 12.8 (p = 0.001) and the synovitis score in PDUS from 10.6 to 4.1 (p < 0.001). The erosion score decreased from 21.5 to 18.1 (p = 0.046). There were longitudinal significant correlations between the new erosion score and both the DAS28 (r = 0.368; p = 0.025) and the synovitis score in PDUS (r = 0.365; p = 0.026) over a 1-year follow-up period. The new erosion score might be a useful tool for the evaluation of erosive changes by US in RA patients. In the course of DMARD and biologic therapy, it was responsive under 1-year follow-up examination.

Usefulness of power Doppler ultrasound for prediction of re-therapy with rituximab in rheumatoid arthritis: a prospective study of longstanding rheumatoid arthritis patients.

OBJECTIVE: To assess the value of gray-scale (GS) and power Doppler (PD) ultrasound (US) in detecting inflammatory/destructive changes and for prediction of necessity of re-therapy with rituximab (RTX) in patients with rheumatoid arthritis (RA) over 1 year of followup. METHODS: GSUS and PDUS were performed to assess synovitis, tenosynovitis, and erosions on the clinically dominant hand and forefoot of 20 patients with RA before and after therapy with RTX. US parameters were compared with clinical (Disease Activity Score in 28 joints, tender/swollen joint counts, and patients' visual analog scale of disease activity) and laboratory parameters (C-reactive protein level and erythrocyte sedimentation rate). Results were compared for patients with and without re-therapy with RTX. RESULTS: Significant decreases in clinical and laboratory parameters were observed after 6 and 12 months. US synovitis scores significantly decreased after 6 and 12 months (P < 0.05 for each). Regarding patients who received re-therapy between 6 and 9 months after the start of therapy (n = 9), a fair therapy response was still detectable before re-therapy. In these patients, PD-positive synovitis was the only parameter that increased up to the 6-month examination. All patients negative for rheumatoid factor and anti-cyclic citrullinated peptide (n = 4) were in the group of patients receiving a second course of treatment. Seropositive patients showed a better response to treatment with less need for re-therapy. CONCLUSION: Response to therapy was measurable by clinical and laboratory parameters as well as by US. Since PDUS was able to detect the onset of disease activity before worsening of clinical symptoms occurred, PDUS is most helpful in evaluating disease activity and making earlier therapy decisions.

Detailed Joint Region Analysis of the 7-Joint Ultrasound Score: Evaluation of an Arthritis Patient Cohort over One Year.

Objective. The main objective of this study was to evaluate the 7-joint ultrasound (US7) score by detailed joint region analysis of an arthritis patient cohort. Methods. The US7 score examines the clinically most affected wrist, MCP and PIP II, III, MTP II, and V joints for synovitis, tenosynovitis/paratenonitis, and erosions. Forty-five patients with rheumatoid arthritis (RA) (84.4%) and spondyloarthritis with polyarticular peripheral arthritis (PsA 13.3%; AS 2.2%) with a median disease duration of 6.5 yrs (range 7.5 mths-47.6 yrs) were included and examined at baseline and 3, 6, and 12 months after starting or changing therapy (DMARD/biologic). In this study, detailed US7 score joint region analysis was firstly performed. Results. The joint region analysis performed at baseline disclosed synovitis in 95.6% of affected wrists in the dorsal aspect by greyscale (GS) US where Grade 2 (moderate) was most often (48.9%) detected. Palmar wrist regions presented Grade 1 (minor) capsule elevation in 40% and Grade 2 (moderate synovitis) in 37.8%. Tenosynovitis of the extensor carpi ulnaris (ECU) tendon was found in 40%, with PD activity in 6.6%. Most of the erosions in MCP II were detected in the radial (68.9%), followed by the dorsal (48.9%) and palmar (44.4%) aspects. In MTP V, erosions were seen in 75.6% from lateral. Conclusions. Synovitis in GSUS was more often detected in the wrist in the dorsal than in the palmar aspect. ECU tendon involvement was frequent. Most erosions were found in the lateral scan of MTP V and the medial (radial) scan of MCP II.

Reliability of the novel 7-joint ultrasound score: results from an inter- and intraobserver study performed by rheumatologists.

OBJECTIVE: To assess the inter- and intraobserver reliability of 26 rheumatologists when performing the 7-joint ultrasound score (US7). METHODS: Six patients with rheumatoid arthritis were examined by 26 sonographers in 12 rater groups who performed the US7 score. The US7 score includes the clinically dominant wrist, the second and third metacarpophalangeal (MCP) and proximal interphalangeal joints, and the second and fifth metatarsophalangeal (MTP) joints, which were evaluated for synovitis, tenosynovitis/paratenonitis, and erosions from the dorsal side and palmar/plantar aspects by gray-scale and power Doppler (PD) ultrasound. Additional lateral scans were performed at the MCP2 and MTP5 joints. All of the groups repeated the examination in 4 patients in order to calculate the intraobserver reliability. The results of one group that included 2 expert sonographers were considered as the reference standard. Kappa values, median agreement rates (interobserver), and P values (intraobserver evaluation) were calculated. RESULTS: The median overall kappa value for detecting synovitis was 0.51, for tenosynovitis/paratenonitis was 0.57, and for erosions was 0.45. In detail, the best interobserver results were found for the detection of erosions in the MTP2 joint from the plantar aspect (κ = 1; median agreement rate 89.4%) and for PD signal detection in the palmar wrist region (κ = 0.79; median agreement rate 78.8%). Good agreement was found for detecting erosions in the MCP2 joint from the radial side (κ = 0.67; median agreement rate 77.3%). CONCLUSION: The inter- and intraobserver reliability of the US7 score shows moderate to substantial kappa values and good agreements. Therefore, this ultrasound score has the potential to be an important imaging tool, including multicenter analysis to assess structural changes.

Characterization of the lactose-specific enzymes of the phosphotransferase system in Lactococcus lactis.

The plasmid-encoded lactose genes of the Lactococcus lactis phosphotransferase system encoding Enzyme IIIlac (lacF) and Enzyme IIlac (lacE) have been identified and cloned in Escherichia coli and L. lactis. Nucleotide sequence and transcription analysis showed that these genes are organized into a lactose-inducible operon with the gene order lacF-lacE-lacG-lacX, the latter two genes encoding phospho-beta-galactosidase and a 34-kDa protein with an unknown function, respectively. The lac-operon is immediately followed by an IS element that is homologous to ISS1. Enzyme IIIlac was purified from L. lactis and determination of its NH2-terminal sequence demonstrated that the lacF gene starts with a TTG codon and encodes a 105 amino acid protein (Mr = 11416). Cross-linking studies with the purified enzyme showed that Enzyme IIIlac is active as a trimer. A mutant lacF gene was identified in strain YP2-5 and appeared to encode Enzyme IIIlac containing the missense mutation G18E. The lacF gene could be expressed under control of vector-located promoter sequences resulting in overproduction of Enzyme IIIlac in E. coli and complementation of the L. lactis lacF mutant YP2-5. The deduced amino acid sequence of Enzyme IIlac consists of 586 amino acids (Mr = 61562) and shows the characteristics of a hydrophobic, integral membrane protein. The deduced primary structures of the L. lactis Enzyme IIIlac and Enzyme IIlac are homologous to those of Staphylococcus aureus (72 and 71% identity, respectively) and Lactobacillus casei (48 and 47% identity, respectively). In contrast, the organization of the lactose genes differs significantly between those Gram-positive bacteria. Heterogramic homology in specific domains was observed between the derived amino acid sequences of the lactose-specific enzymes and that of E. coli Enzyme IIIcel and Enzyme IIcel, which suggest a common function in the transport and phosphorylation of these structurally related beta-glucosides.

Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus.

The gene which encodes the mannitol-specific enzyme III (EIIImtl) of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus, has been cloned. Genomic libraries of S. carnosus DNA were constructed using the expression vector pUC19 and EIIImtl-producing clones were identified using rabbit polyclonal antiserum. A 700-bp Dde I fragment, containing the complete gene encoding EIIImtl, was sequenced by the dideoxy chain-termination technique. Upstream from the ORF for EIIImtl one can find a sequence analogous to that of the Escherichia coli promoter. This region acts as a strong promoter when subcloned into the promoter test vector M13HDL17. EIIImtl was overproduced using the inducible T7 polymerase system and purified to homogeneity. Amino acid sequence comparison confirmed a 38% similarity to the hydrophilic enzyme-III-like portion of enzyme IImtl of E. coli. There is also a 36% similarity to the N terminus of the fructose-specific phospho-carrier protein from E. coli.

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli.

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.

Characterization of mutant histidine-containing proteins of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium.

Histidine-containing phosphocarrier protein (HPr) is common to all of the phosphoenolpyruvate:sugar phosphotransferase systems (PTS) in Escherichia coli and Salmonella typhimurium, except the fructose-specific PTS. Strains which lack HPr activity (ptsH) have been characterized in the past, and it has proved difficult to delineate between tight and leaky mutants. In this study four different parameters of ptsH strains were measured: in vitro sugar phosphorylation activity of the mutant HPr; detection of 32P-labeled P-HPr; ability of monoclonal antibodies to bind mutant HPr; and sensitivity of ptsH strains to fosfomycin. Tight ptsH strains could be defined; they were fosfomycin resistant and produced no HPr protein or completely inactive mutant HPr. All leaky ptsH strains were fosfomycin sensitive, usually produced normal amounts of mutant HPr protein, and had low but measurable activity, and HPr was detectable as a phosphoprotein. This indicates that the regulatory functions of the PTS require a very low level of HPr activity (about 1%). The antibodies used to detect mutant HPr in crude extracts were two monoclonal immunoglobulin G antibodies Jel42 and Jel44. Both antibodies, which have different pIs, inhibited PTS sugar phosphorylation assays, but the antibody-HPr complex could still be phosphorylated by enzyme I. Preliminary evidence suggests that the antibodies bind to two different epitopes which are in part located in a beta-sheet structure.