Autoimmune responses against posttranslationally modified antigens are a hallmark of several autoimmune diseases. For example, antibodies against citrullinated protein antigens (ACPA) have shown their relevance for the prognosis and diagnosis of rheumatoid arthritis (RA), and have been implicated in disease pathogenesis. It is conceivable that other autoantibody systems, recognizing other posttranslationally modified proteins, are also present in RA. Here, we describe the presence of an autoantibody system that discriminates between citrulline- and homocitrulline-containing antigens in the sera of RA-patients. IgG antibodies recognizing carbamylated (homocitrulline-containing) antigens were present in sera of over 45% of RA-patients. Likewise, anticarbamylated protein (anti-CarP) IgA antibodies were observed in 43% of RA-sera. ACPA and anti-CarP antibodies are distinct autoantibodies because, in selected double-positive patients, the anti-CarP antibody binding to carbamylated antigens could be inhibited by carbamylated antigens, but not by control or citrullinated antigens. Similarly, ACPA-binding to citrullinated antigens could only be inhibited by citrullinated antigens. In line with this observation, 16% of ACPA-negative RA-patients, as measured by a standard ACPA assay, harbored IgG anti-CarP antibodies, whereas 30% of these patients tested positive for IgA anti-CarP antibodies. The presence of anti-CarP antibodies was predictive for a more severe disease course in ACPA-negative patients as measured by radiological progression. Taken together, these data show the presence of a unique autoantibody system recognizing carbamylated, but not citrullinated, protein antigens. These antibodies are predictive for a more severe clinical course in ACPA-negative RA-patients, indicating that anti-CarP antibodies are a unique and relevant serological marker for ACPA-negative RA.

Objective. Rheumatoid arthritis (RA) is characterized by inflammation and joint destruction, with the degree of damage varying greatly among patients. Prediction of disease severity using known clinical and serologic risk factors is inaccurate. This study was undertaken to identify new serologic markers for RA severity using an in silico model of the rheumatic joint.Methods. An in silico model of a prototypical rheumatic joint was used to predict candidate markers associated with erosiveness. The following 4 markers were chosen for validation: tartrate-resistant acid phosphatase 5b (TRAP-5b), N-telopeptide of type I collagen (NTX), angiopoietin 2 (Ang-2), and CXCL13. Serum from 74 RA patients was used to study whether radiologic joint destruction (total erosion score and total Sharp/van der Heijde score [SHS]) after 4 years of disease was associated with serum levels at the time of diagnosis. Serum marker levels were determined using enzyme-linked immunosorbent assays. For confirmation, baseline serum levels were analyzed for an association with progression of joint damage over 7 years of followup in a cohort of 155 patients with early RA.Results. Comparison of high and low quartiles of erosion score and SHS at 4 years showed a difference in baseline serum CXCL13 level (P ‫؍‬ 0.011 and P ‫؍‬ 0.018, respectively). In the confirmation cohort, elevated baseline CXCL13 levels were associated with increased rates of joint destruction during 7 years of followup (P < 0.001 unadjusted and P < 0.004 with adjustment for C-reactive protein level). Analyzing anti-CCP-2-positive and anti-CCP-2-negative RA separately yielded a significant result only in the anti-CCP-2-negative group (P < 0.001).Conclusion. Our findings indicate that CXCL13 is a novel serologic marker predictive of RA severity. This marker was identified with the help of an in silico model of the RA joint.

The effect of a brief beta-lactam action on a bacterial culture has been studied by combining the use of a high performance photometer with phase contrast microscopic examination. After exposure of the culture with the antibiotic, the growth curves presented in logn OD have shown the importance of the pre-lytic increase in OD (PIOD). Using Escherichia coli a study of the PIOD values allowed us to separate the beta-lactam antibiotics studied into two groups: those whose PIOD were concentration-dependent (ampicillin, carbenicillin, ticarcillin, moxalactam, ceftazidime, cefsulodin, cefotaxime) and those whose PIOD were concentration-independent (or weakly dependent) (azlocillin, mezlocillin, piperacillin). When the PIOD was independent of the concentrations, long filaments were observed by phase contrast microscopy. Using Pseudomonas aeruginosa, all the antibiotics studied produced PIOD values that were barely dependent of the concentrations, and long filaments were observed by phase contrast microscopy. Study of the lag of regrowth in the post antibiotic period using a beta-lactamase technique showed that, if the PIOD was hardly concentration dependent, so was the lag of regrowth.

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Urinary tract infections caused by P-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.Formulations of amoxicillin with potassium clavulanate possess activity against many plasmid-mediated P-lactamase-producing bacteria (3, 4, 7-11, 13-15, 19) and have been used successfully in the therapy of urinary tract infections (1,8).Two formulations were assessed in this trial, which included 22 adult in-patients (17 women and 5 men, ranging in age from 50 to 90 years) suffering from 24 episodes of urinary tract infection caused by amoxicillin-resistant organisms. Ten episodes (9 patients, group 1) were treated for 10 days with the 2:1 combination (500 mg of amoxicillin plus 250 mg of clavulanic acid administered three times daily), and fourteen episodes (13 patients, group 2) were treated with the 4:1 combination (500 mg of amoxicillin plus 125 mg of clavulanic acid administered three times daily). Tablets were taken with food. Exclusion criteria were allergy to P-lactams, renal impairment (creatinine clearance, <30 ml/ min), hepatic failure, bleeding disorders, bladder catheterization, recent administration of an effective antibiotic, and infection with an organism resistant to combinations of amoxicillin and clavulanic acid. Complete physical examinations were obtained before, during, and after treatment. Complete blood counts with differential leukocyte counts, platelets counts, hemoglobin levels, and concentrations of creatinine, urea, alkaline phosphatase, transaminases, and bilirubin in serum were also made at these times. "Clean-catch" urine was considered infected if yielding i105 organisms per ml on at least two separate occasions, together with significant pyuria (8). Clinical pyelonephritis was confirmed by the presence of antibody-coated bacteria (18). Antibiotic susceptibility was determined by an agar diffusion method (2), using filter-paper disks impregnated with amoxicillin alone (20 ,ug) and in combination with clavulanic acid (20 ,ug of amoxicillin and 10 ,ug of clavulanic acid), both kindly provided by Beecham Laboratories. Bacteria were considered susceptible if the diameter of the growth inhibition zone was >'19 mm and resistant if the zone was -14 mm. Minimal inhibitory concentrations (MICs) were determined by an agar dilution method. A multiinoculator (16) was used to deliver 4 x 103 colonyforming units per spot on the surfaces of Mueller-Hinton agar plates containing twofold serial dilutions of the combination over a suitable concentration range. The stock solution (300 ,ug/ml), prepared from pellets (provided by Beecham Laboratories) containing 2 mg of amoxicillin and 1 mg of clavulanic acid, was diluted in distilled water. The breakpoints were identical for amoxicillin alone and for the drug combination; i.e.,...

Escherichia coli strains that were susceptible to multiple antibiotics were exposed to suprainhibitory concentrations of ampicillin and piperacillin. As with the majority of beta-lactam antibiotics, the growth curves showed an increase in optical density (OD) before lysis during the first hours. This increase in OD depended on the concentration of ampicillin and was independent of the concentration of piperacillin. A good correlation was found between the prelytic increase in OD and the killing curve. During the prelytic increase in OD, the number of CFU per milliliter remained constant. The decrease in the number of CFU per milliliter depended on the concentration of ampicillin and was independent of the concentration of piperacillin. pH variations gave rise to similar effects on growth curves and killing curves.The antimicrobial activity of antibiotics is usually defined by the MIC and the MBC. This usually involves a single value obtained after one night of incubation. Growth curves allow an approach which documents the effect of a brief contact of an antibiotic with a bacterial culture (4). However, growth curves must be interpreted with great caution. Indeed, an increase or a decrease in optical density (OD) may be due to very different microbiological effects. Our previous study (12) showed the value of measuring the prelytic increase in OD. During the first 2 or 3 h after the introduction of beta-lactam antibiotics into an Escherichia coli culture, an increase in OD which could be superimposed on the control curve was observed. This increase in OD was due to the formation of cells with deficient cell walls.The aim of the present study was (i) to verify whether bacteria remain viable during the prelytic increase in OD and, if so, in what proportion, and (ii) to determine whether variations in culture media (e.g., of pH) influence growth curves and the number of CFU per milliliter in the same way. Since it was a matter of verifying the agreement between growth curves and killing curves, we limited our study to strains of E. coli that were susceptible to multiple antibiotics. Only the early response (the first 6 h of contact between the antibiotic and the culture) were taken into consideration. We chose ampicillin and piperacillin at three suprainhibitory concentrations, 16, 64, and 256 ,ug/ml. Indeed, our previous study (12) [Oxoid Ltd., London, England]; macrodilution method; inoculum, 106 CFU/ml) was 1.56 ,ug/ml, with a possible variation of one dilution after 24 h of incubation at 35°C.Culture media. Growth curves and killing curves were determined with Iso-Sensitest medium. Modifications of pH were obtained by adding an acid or a base. The osmolarities of the media so modified were 342 mOsm/k at pH 7.3, 441 mOsm/k at pH 6.3, and 433 mOsm/k at pH 8.3. Colony counts were made on the same media containing 1.5% agar.Growth curves. The MS-2 system (Abbott Laboratories, Diagnostics Div., Irving, Tex.) (8) and open multichamber cuvettes were used to determine growth curves. Ampicillin and piperacillin were ...

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.

Ten strains of Pseudomonas aeruginosa that were susceptible to imipenem (MICs 2 mg/l) were exposed to a new parenteral carbapenem, meropenem (MIC 0.25 mg/l). Kinetic turbidometry showed that, as with other beta-lactam antibiotics, there was a prelytic increase in the culture OD following exposure to meropenem. The maximal value of the prelytic increase in the OD was higher for meropenem than for imipenem at concentrations 0.5, 1, 2, 4 and 8 x MIC. This corresponded to the formation of short filaments during exposure to low concentrations of meropenem. These filaments remained viable for 1-2 h, according to the drug concentration. For this reason, the killing began later with meropenem than with imipenem. After this delay, the killing rate for meropenem was the same as with imipenem, but occurred with lower concentrations of meropenem.