A genetic variant in granzyme B is associated with progression of joint destruction in rheumatoid arthritis.

OBJECTIVE: Genetic factors account for an estimated 45-58% of the variance in joint destruction in rheumatoid arthritis (RA). The serine proteinase granzyme B induces target cell apoptosis, and several in vitro studies suggest that granzyme B is involved in apoptosis of chondrocytes. Serum levels of granzyme B are increased in RA and are also associated with radiographic erosions. The aim of this study was to investigate GZMB as a candidate gene accounting for the severity of joint destruction in RA. METHODS: A total of 1,418 patients with 4,885 radiograph sets of the hands and feet from 4 independent cohorts were studied. First, explorative analyses were performed in 600 RA patients in the Leiden Early Arthritis Clinic cohort. Fifteen single-nucleotide polymorphisms (SNPs) tagging GZMB were tested. Significantly associated SNPs were genotyped in data sets representing patients from the Groningen, Sheffield, and Lund cohorts. In each data set, the relative increase in the annual rate of progression in the presence of a genotype was assessed. Data were summarized in a meta-analysis. The association of GZMB with the RNA expression level of the GZMB genomic region was tested by mapping expression quantitative trait loci (QTLs) on 1,469 whole blood samples. RESULTS: SNP rs8192916 was significantly associated with the rate of joint destruction in the first cohort and in the meta-analysis of all data sets. Patients homozygous for the minor allele of rs8192916 had a higher rate of joint destruction per year compared with other patients (P = 7.8 × 10(-4)). Expression QTL of GZMB identified higher expression in the presence of the minor allele of rs8192916 (P = 2.27 × 10(-5)). CONCLUSION: SNP rs8192916 located in GZMB is associated with the progression of joint destruction in RA as well as with RNA expression in whole blood.

Comparison of methodologies for analysing the progression of joint destruction in rheumatoid arthritis.

OBJECTIVES: Progression of joint destruction is an important phenotypic feature in rheumatoid arthritis (RA). When factors have small effect sizes, both the avoidance of phenotypic misclassification and discerning true effects from noise are challenging. Assembling radiological measurements repeatedly in time harbours a smaller risk of misclassification than single measurements. Given serial measurements, different methods of analysis can be applied. This study evaluates different statistical methods of analysing longitudinal data. METHODS: Three statistical methods were studied: linear regression (LR), generalized estimating equations (GEE), and multivariate normal regression analysis (MRA). All were applied longitudinally, testing for differences in radiological progression rates. As genetic variants are known to have small effect sizes, two genetic variants were studied as examples: rs675520 (located in the TNFAIP3-OLIG3 region) and the presence of the human leucocyte antigen (HLA) shared epitope (SE) alleles. Radiological data for 602 early RA patients with yearly radiographs and 7-years of follow-up were used. The powers obtained with the methods and the robustness against missingness were evaluated as outcome measures. RESULTS: The presence of the rs675520 polymorphism and the HLA-SE risk genotype was associated with a 0.65-0.77 and 1.17-1.51 fold increased rate of joint destruction, respectively. The analyses performed with MRA resulted in smaller 95% confidence intervals (CIs) than the analyses using LR or GEE. In addition, the 95% CIs increased with the number of radiographs per patient. The power of MRA was higher than that of GEE. MRA was more robust against selective missingness than GEE or LR with a two-step approach (LR(ts)). CONCLUSIONS: A multivariate normal regression model on subsequent radiographs is a powerful and robust method for analysing longitudinal joint destruction data.

Genetic variants in IL15 associate with progression of joint destruction in rheumatoid arthritis: a multicohort study.

BACKGROUND: Interleukin (IL)-15 levels are increased in serum, synovium and bone marrow of patients with rheumatoid arthritis (RA). IL-15 influences both the innate and the adaptive immune response; its major role is activation and proliferation of T cells. There are also emerging data that IL-15 affects osteoclastogenesis. The authors investigated the association of genetic variants in IL15 with the rate of joint destruction in RA. METHOD: 1418 patients with 4885 x-ray sets of both hands and feet of four independent data sets were studied. First, explorative analyses were performed on 600 patients with early RA enrolled in the Leiden Early Arthritis Clinic. Twenty-five single-nucleotide polymorphisms (SNPs) tagging IL-15 were tested. Second, SNPs with significant associations in the explorative phase were genotyped in data sets from Groningen, Sheffield and Lund. In each data set, the relative increase of the progression rate per year in the presence of a genotype was assessed. Subsequently, data were summarised in an inverse weighting meta-analysis. RESULTS: Five SNPs were significantly associated with rate of joint destruction in phase 1 and typed in the other data sets. Patients homozygous for rs7667746, rs7665842, rs2322182, rs6821171 and rs4371699 had respectively 0.94-, 1.04-, 1.09-, 1.09- and 1.09-fold rate of joint destruction compared to other patients (p=4.0×10(-6), p=3.8×10(-4), p=5.0×10(-3), p=5.0×10(-3) and p=9.4×10(-3)). DISCUSSION: Independent replication was not obtained, possibly due to insufficient power. Meta-analyses of all data sets combined resulted in significant results for four SNPs (rs7667746, p<0.001; rs7665842, p<0.001; rs4371699, p=0.01; rs6821171, p=0.01). These SNPs were also significant after correction for multiple testing. CONCLUSION: Genetic variants in IL-15 are associated with progression of joint destruction in RA.

Classification of rheumatoid arthritis: comparison of the 1987 American College of Rheumatology criteria and the 2010 American College of Rheumatology/European League Against Rheumatism criteria.

OBJECTIVE: New criteria to classify rheumatoid arthritis (RA) have been derived in order to increase the specificity and sensitivity for early RA compared with the 1987 American College of Rheumatology (ACR) criteria. The aim of this study was to evaluate differences in classification between the 1987 ACR criteria and the 2010 ACR/European League Against Rheumatism (EULAR) criteria in an early arthritis cohort and to determine the test characteristics of the 2010 ACR/EULAR criteria. METHODS: A total of 2,258 patients with early arthritis included in the Leiden Early Arthritis Clinic cohort were studied. Fulfillment of the 1987 and 2010 criteria for the classification of RA was determined at baseline. The diagnosis of each patient at 1 year was assessed. The sensitivity and specificity of the 2010 criteria were determined using the following outcome measures: initiation of methotrexate therapy or any disease-modifying antirheumatic drug (DMARD) therapy during the first year of followup and having persistent arthritis during 5 years of followup. RESULTS: At their first presentation, 1,099 patients fulfilled the 2010 criteria, and 726 patients fulfilled the 1987 criteria for RA. Eighty-two of the 726 patients fulfilling the 1987 criteria did not fulfill the 2010 criteria. Sixty-eight percent of the patients who fulfilled the 1987 criteria during the first year of disease but not at baseline did fulfill the 2010 criteria at baseline. In 18% of patients, use of the 2010 classification criteria also led to a revoked classification at 1 year. The sensitivity and specificity of the 2010 criteria were 0.84 and 0.60, respectively, with methotrexate therapy as the outcome and 0.74 and 0.74, respectively, with DMARD therapy as the outcome. With persistent arthritis as the outcome, the sensitivity and specificity of the 2010 criteria were 0.71 and 0.65, respectively. CONCLUSION: Compared with the 1987 criteria, the 2010 criteria classify more patients with RA and at an earlier phase of the disease. The discriminative ability of the 2010 criteria is acceptable.

Toward a data-driven evaluation of the 2010 American College of Rheumatology/European League Against Rheumatism criteria for rheumatoid arthritis: is it sensible to look at levels of rheumatoid factor?

OBJECTIVE: Recently, new classification criteria for rheumatoid arthritis (RA) have been devised by methodology that used first a quantitative approach (data from databases), then a qualitative approach (consensus; based on paper patients), and finally a common sense-based approach (evaluation of the former phases). Now the individual items that make up these criteria are being evaluated. This study was undertaken to analyze the item "autoantibodies," in particular rheumatoid factor (RF) level. METHODS: Three separate cohorts comprising a total of 972 patients with undifferentiated arthritis were studied for RA development (according to the 1987 American College of Rheumatology criteria) and arthritis persistence. The positive predictive value (PPV), negative predictive value (NPV), and likelihood ratios (LRs) were compared between different levels of RF and the presence of anti-citrullinated protein antibody (ACPA). A similar comparison was made in 686 RA patients for the rate of joint destruction and achievement of sustained disease-modifying antirheumatic drug-free remission during 7 years of followup. The variation in RF levels obtained by different measurement methods in the same RF-positive sera was explored. RESULTS: Compared to high RF levels, presence of ACPA had a better balance between positive LR and negative LR and between PPV and NPV for RA development. The additive value of ACPA assessment after testing for RF level was higher than vice versa. The association between high RF level and RA severity was not as strong as that between ACPA antibodies and RA severity. The RF level obtained by different methods in the same patients' sera varied considerably. CONCLUSION: Our findings indicate that determination of RF level is subject to large variation; high RF level has limited additive prognostic value compared to ACPA positivity. Thus, omitting RF level and using RF presence, ACPA presence, and ACPA level may improve the 2010 criteria for RA.

Use and interpretation of Schlichter's test on Haemophilus influenzae: relation of in vitro to in vivo results for cefamandole.

When Haemophilus influenzae infections are treated by an antibiotic acting on the bacterial wall, the adequacy of antimicrobial therapy can be assessed by Schlichter's test. This test may be carried out using Mueller Hinton broth (or Mueller Hinton broth with 50% pooled serum and a supplement of Ca++ and Mg++) supplemented with Fildes' enrichment and an inoculum adjusted to the 0.5 McFarland turbidity standard diluted 200x. However, correct reading of end points can be obtained only by phase contrast microscopic examination, which allows the establishment of good correlation between the in vitro and in vivo findings. In patients with lung infections successfully treated with cefamandole, the presence of spheroplasts in samples derived from Schlichter's tests correlates well with clinical improvement and eradication of the pathogenic organism checked by transtracheal aspiration.

Combination of minocycline and rifampicin against methicillin- and gentamicin-resistant Staphylococcus aureus.

Methicillin- and gentamicin-resistant Staphylococcus aureus may remain sensitive to minocycline and to rifampicin. A study of growth curves has shown that at inhibitory concentrations (0.4 mug/ml), minocycline prevents the development of mutants resistant to rifampicin.

In vivo crystal formation in Escherichia coli of an over-expressed soluble form of penicillin-binding protein 5.

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in the cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped crystal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultrastructure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, gamma = 75 degrees. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.

Growth curve patterns of Escherichia coli, Serratia marcescens, and Proteus vulgaris submitted to different tigemonam concentrations.

Five strains of Escherichia coli, Serratia marcescens, and Proteus vulgaris were exposed to a new monobactam, tigemonam, in comparison with aztreonam. The study, evaluated by kinetic turbidimetry, has shown that tigemonam exerts a prelytic increase in optical density (OD) similar to that of other beta-lactam antibiotics. The maximal value of the prelytic increase in OD was similar for the two study antibiotics at concentrations of 1, 2, 4, and 8 times the minimum inhibitory concentration, corresponding with filament formation. The OD values varied according to the bacterial species. Bactericidal activity was observed for the three species evaluated. At 6 hours, the killing rate was not dose-related. In conclusion, tigemonam and aztreonam induced first filament formation followed after a few hours by bactericidal activity.

Antibacterial effects of meropenem and imipenem against Haemophilus influenzae.

Phase contrast microscopy, killing curves and turbidimetric growth curves were used in a comparative study of the antibacterial effects of imipenem and meropenem on Haemophilus influenzae. The minimal inhibitory concentrations (MICs) and their ranges of meropenem and imipenem using five beta-lactamase-producing strains of H. influenzae were 0.03 (0.015-0.06) and 0.6 (0.5-1) micrograms/ml, respectively. Imipenem and meropenem induced spheroplast formation in cultures. Killing curves showed a bacteriostatic activity for meropenem and imipenem for MIC values, and a lag of 2 h in killing for MIC x 2 to MIC x 64. For these concentrations the killing rates of the two antibiotics were similar. Turbidimetric growth curves showed a higher early increase in optical density for meropenem. As far as the MIC value of meropenem was 10 times lower than the MIC value of imipenem, we may conclude that meropenem was more active than imipenem on beta-lactamase-producing strains of H. influenzae.

Correlation between growth curves and killing curves of Escherichia coli in the presence of fleroxacin and ampicillin.

Fleroxacin, a new long-acting quinolone, induces rapid killing and bacterial filamentation as do other quinolones. Ten strains of Escherichia coli were exposed comparatively to fleroxacin and ampicillin in order to determine the effect of sub- and supra-inhibitory concentrations of each of these two compounds on turbidimetric growth curves and viable counts. By comparing the maximal early increase in optical density (OD, PIOD) as colony-forming units per milliliter (CFU/ml) after 2 and 6 h of exposure to antibiotics, we observed a reduced number of CFU/ml in comparison with the control after the 2-hour exposure at 1/4 the minimum inhibition concentration (MIC) and after 6 h at 1/8 MIC, but a high OD value was also seen among the fleroxacin exposed bacteria. For ampicillin, PIOD rates and killing rates were slower and dose dependent. This discrepancy was due to filament formation, which increased the PIOD value to the same extent as the control curve. After exposure to fleroxacin at 1/2 MIC the PIOD decreased significantly and after 2 and 6 h E. coli killing rates of 99 and 99.9%, respectively, were observed. With exposure to 2 and 4 x MIC, both PIOD values and CFU/ml decreased substantially. Combined analysis of continuous turbidimetric monitoring and viable counts showed that subinhibitory concentrations of fleroxacin and beta-lactam had different effects on E. coli. Fleroxacin's rapid killing rate, despite filament formation, contrasted with the result obtained with ampicillin. The minimum antibiotic concentration of fleroxacin against E. coli was around 1/8 MIC.

Killing rate and growth rate comparison for newer beta-lactamase-stable oral beta-lactams against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.

A new method of data presentation that takes into account the relationship between growth and killing rate was used to evaluate the comparative in vitro bactericidal activity of cefpodoxime, cefuroxime, cefixime and an amoxicillin/clavulanic acid combination against Streptococcus pneumoniae and beta-lactamase-producing strains of Haemophilus influenzae and Moraxella catarrhalis. For each strain, the viable count decrease (log CFU/ml) after 6 h of exposure to different antibiotic concentrations was plotted against the viable count increase in the control culture, over the same time. Higher killing rates than those predicted by growth rates were defined as a positive balance; lower rates than those predicted by growth rates were defined as a negative balance. The activity of the 4 drugs against S. pneumoniae and M. catarrhalis was characterized by a positive balance. Conversely, the 3 cephalosporins showed a negative balance for H. influenzae.

Comparative kill and growth rates determined with cefdinir and cefaclor and with Streptococcus pneumoniae and beta-lactamase-producing Haemophilus influenzae.

The relationship between the growth rate and the kill rate was used to evaluate and to compare the in vitro bactericidal activities of cefdinir, a new oral cephalosporin, and cefaclor against Streptococcus pneumoniae and beta-lactamase-producing strains of Haemophilus influenzae. These frequently encountered pathogens of community-acquired respiratory tract infections are usually susceptible to both drugs. The MIC ranges for cefdinir and cefaclor were, respectively, 0.03 to 0.06 and 0.25 to 0.5 micrograms/ml for S. pneumoniae and 0.25 and 4 to 8 micrograms/ml for H. influenzae. The colony counts (CFU per milliliter) measured after 6 h of exposure to a range of antibiotic concentrations in broth were plotted against the colony count of the control culture over the same period of time. Higher kill rates versus bacterial growth rates were noted for S. pneumoniae for both drugs (positive balance). Conversely, lower kill rates versus growth rates were noted for H. influenzae for both drugs (negative balance). In conclusion, the bactericidal activities of both drugs against S. pneumoniae and H. influenzae were similar when expressed by the relationship between the growth rate and the kill rate at 6 h, but cefdinir was more active at lower concentrations.

Cytoplasmic high-level expression of a soluble, enzymatically active form of the Escherichia coli penicillin-binding protein 5 and purification by dye chromatography.

High-level expression of a soluble form of penicillin-binding protein 5 (PBP5), called PBP5s, and translocation across the cytoplasmic membrane results in lysis of Escherichia coli cells. The detrimental effect of increased amounts of this D,D-carboxypeptidase on the stability of murein polymer can be avoided by accumulation of the overexpressed protein in the cytoplasm. The signal peptide of the structural gene dacAs, coding for PBP5s was deleted by creating a BamHI site at the site of processing and the truncated gene dacAsc was cloned under the control of the lambda PR promoter. Temperature induction resulted in a 200-fold overproduction of the mature PBP5s in the cytosol (PBP5sc) which is no longer harmful to the cells. PBP5sc could quantitatively be recovered in the soluble fraction after disrupting the cells. The protein retained full enzymatic activity as measured by the release of D-alanine from bisacetyl-L-Lys-D-Ala-D-Ala and formation of [14C]penicillin-protein complex at a 1:1 stoichiometry. A one-step purification procedure using the immobilized dye Procion rubine MX-B resulted in homogeneous preparations of both wild-type and mutated forms of PBP5sc.

Activities of amoxicillin and clavulanic acid combinations against urinary tract infections.

Urinary tract infection caused by beta-lactamase-producing bacteria were treated with 500 mg of amoxicillin three times daily plus either 250 or 125 mg of clavulanic acid three times daily. The overall cure rate was 63.6%, 77.7% with the high dose of clavulanic acid and 53.8% with the low dose. Gastrointestinal intolerance was common in the high-dose clavulanic acid regimen.

Antibacterial effect of meropenem and imipenem on Proteus mirabilis.

Phase-contrast microscopy, killing-curves and turbidimetric growth-curves were used in a comparative study of the antibacterial effects of a new carbapenem, meropenem (SM 7338) and imipenem on five strains of Proteus mirabilis. Despite the low MIC (0.2 mg/l) of imipenem for the five strains included in our study, the MBC remained relatively high (4.4 mg/l). During the first few hours of incubation, imipenem induced large lemon-shaped cells while the turbidity increased without substantial changes in culture viability. Later, most of the cell-wall deficient bacteria generated small spheroplasts until the antibiotic concentration exceeded 32 times the MIC. The MIC of meropenem was lower (0.03 mg/l) with an MBC (0.08 mg/l) very close to the MIC. Meropenem also induced large bodies but these cell-wall deficient bacteria did not generate small round bodies as observed with imipenem. In conclusion, imipenem produced in strains of Pr. mirabilis an amdinocillin-like change in cell morphology, responsible for the discrepancies observed between MIC and MBC. This effect was not observed with meropenem.

Inoculum effect on growth-delay time of oxacillin-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis exposed to cefamandole, cefazolin, and cefuroxime.

Cephalosporins have been recommended as prophylactic antibiotics in patients undergoing cardiovascular surgery. The major function of these antibiotics is to protect patients against Staphylococcus aureus and Staphylococcus epidermidis infections. The lowest inoculum amount responsible for infection during surgery is unknown but is probably low. To determine the comparative activities of cefazolin, cefuroxime, and cefamandole against S. aureus and S. epidermidis for prophylactic purposes, we selected five strains of S. aureus and S. epidermidis that presented homogeneous resistances to oxacillin. A continuously monitored turbidimetric method was used to evaluate cultures with variable inoculum sizes ranging from 10(6) to 1 CFU/ml and exposed to cefazolin, cefuroxime, and cefamandole at concentrations of 0.5, 1, 2, 4, 8, 16, and 32 micrograms/ml. Growth was defined as an increase of 0.1 optical density unit. The relationship between the time required for growth, the antibiotic concentration, and the initial bacterial density showed that cefamandole was more active than cefazolin, which, in turn, was revealed to be more active than cefuroxime against S. aureus and S. epidermidis.

One shot of high-dose amikacin: a working hypothesis.

Recent information suggests that single, large daily dosages of amikacin are less nephrotoxic. The killing rate of amikacin for Escherichia coli and Pseudomonas aeruginosa also suggests to put emphasis on a high peak value. A decrease of 3 log10 CFU/ml was observed for E. coli and P. aeruginosa at 64 and 128 micrograms/ml in 20 min. In comparison, the killing rate of piperacillin was dose-independent and about 6 h were required for a reduction of 10(3) CFU/ml of P. aeruginosa. In theory, the way to proceed in the future would possibly be the one-shot administration of amikacin, followed by a long course of a beta-lactam antibiotic.