Heterogeneity of Fecal Calprotectin Reflecting Generation of Neutrophil Extracellular Traps (NETs) in the Gut: New Immunoassays Are Available

Background: We aimed at obtaining more information on the structure of fecal calprotectin (CP) as a basis for establishing improved quantitative assays and detection of Neutrophil Extracellular Traps (NETs) in stools. Commercial fecal CP assays produce different results, probably due to differences in antibodies, extraction procedures, and standards used. In addition, the structure of fecal CP may be different from that in the standard so that rules for immunoassays are violated. We aimed at solving these problems by studying the structure of fecal CP and developing new antibodies and assay procedures including some for NETs in stools. Methods and Findings: Stool samples from children with abdominal symptoms were extracted by a conventional and a new procedure. Some extracts were run on anion exchange and size exclusion chromatography, and fractions were tested on ELISAs by use of ten new mouse monoclonal antibodies against the CP subunit S100A9. Hybrid ELISAs (named HELISA) were established using anti-DNA or anti-histones for coating of microwells, and enzyme labelled anti-CP was used for development. By ion exchange chromatography, five to ten fecal CP subfraction peaks differing in net electric charge were found, all of which contained the major chromatin components. The presence of DNA and histones followed calprotectin in the chromatographic fractions suggesting that NETs are generated in the gut lumen. The new CP monoclonals reacted very differently against the subfractions so that a mixture of them (called MiMo) must be used to obtain reliable assay values for fecal CP. A new method called FELISA was developed where standards and samples are applied directly in Nunc (Denmark) MaxiSorp plates, without any catching antibody. It takes advantage of the property of CP to bind strongly to the plastic in wells. This method has a higher sensitivity because it will detect CP molecules with only one antigenic epitope available. It will give more reliable estimates and more efficient selection of patients for complex diagnostic procedures. We also developed an alternative to the FELISA: a competitive ELISA where S100A9 coated in microwells will compete with CP in standards and samples for binding to a properly diluted HRP-anti-CP solution. In this method, the presence of other proteins in extraction or dilution buffers will not interfere. Using the HELISA, about 65% of the patients had detectable fecal NETs in concentrations between 150 and 1500 ng/mL; however, the values correlated poorly with CP values. Extraction of fecal samples with a simple buffer of TBS, and pH 5 with 5 mM EDTA, gave a yield of about 90%, while the yields of commercial kits are not specified or lie around 50%. A fecal CP standard will bring methods in accordance with the requirements for immunoassays that the structure of CP in the standard and sample must be the same. A mixture of fecal anion exchange fractions as a standard may be a solution to this problem. The principle worked in the first trial by giving the same values after storage of such a standard at 5C for four months. Conclusions: Fecal CP consists of at least five subfractions containing NETs or degradation products thereof. Commercial kits should not be accepted for clinical use unless it has been shown that they can detect all subfractions which may require the use of a mixture of monoclonals. The methods presented here can be used for such a quality control. The HELISA methods can be used for assays on NETs in stools and to study their possible pathogenic effects in the gut. Use of the FELISA and the S100A9 competitive method may give increased sensitivity, higher precision, and better selection of patients for more complex procedures

The soluble biomarker calprotectin (a S100 protein) is associated to ultrasonographic synovitis scores and is sensitive to change in patients with rheumatoid arthritis treated with adalimumab

Introduction Calprotectin (MRP8/MRP14, S100A8/A9) is associated with disease activity in patients with rheumatoid arthritis (RA). Ultrasonography (US) is a reliable method for evaluation of synovitis (B-mode (BM) and power Doppler (PD)). The present objectives were to explore in RA patients the associations between calprotectin and a comprehensive US examination, as well as the responsiveness of calprotectin compared to other inflammatory markers during anti-TNF treatment. Methods A total of 20 RA patients starting treatment with adalimumab were examined longitudinally by US (BM and PD (semi-quantitative scores 0 to 3) of 78 joints, 36 tendons/tendon groups and 2 bursae) and clinically at baseline and after 1, 3, 6 and 12 months. Associations between the US sum scores and the inflammatory markers calprotectin, serum amyloid A (SAA), CRP and ESR were explored by correlation and linear regression analyses, and the response to treatment was assessed by Standardized Response Mean (SRM). Results The inflammatory markers, clinical examinations and US sum scores improved during treatment (P < 0.001). Of the inflammatory markers, calprotectin had the highest correlation coefficients with the total BM and PD sum scores (median (range) 0.59 (0.37 to 0.76) for BM and 0.56 (0.38 to 0.72) for PD). Even higher correlations were found between calprotectin and sum US scores of reduced number of joint counts. Calprotectin made a considerable contribution to total US sum scores in the linear regression analyses (P = 0.001 to 0.031) and among the inflammatory markers, calprotectin had the highest SRM (0.84 at one month). Conclusions Calprotectin was associated with the sum scores from a comprehensive US assessment and was responsive to change during anti-TNF treatment. Thus, examination of this leukocyte protein could be of additional value in the assessment of RA patients on biologic treatment

Changes in calprotectin concentration in sow’s milk throughout lactation

International audienc

Calprotectin (a major leucocyte protein) is strongly and independently correlated with joint inflammation and damage in rheumatoid arthritis

OBJECTIVE: Calprotectin is a major leucocyte protein, shown to correlate well with laboratory and clinical assessments in several inflammatory rheumatic diseases, and large concentrations of calprotectin have been found in synovial fluid from patients with rheumatoid arthritis (RA). The objective of the present study was to examine correlations between calprotectin and joint damage. METHODS: 145 patients with RA were analysed cross sectionally with laboratory (calprotectin, C reactive protein (CRP), and erythrocyte sedimentation rate (ESR)), clinical (28 joint counts (tender, swollen), physician global VAS, DAS28 and RA Articular Damage score (RAAD)), and radiographic (plain hand radiographs; modified Sharp's method) measurements, on the same day. RESULTS: Calprotectin showed a highly significant correlation with measures of joint damage; modified Sharp score r = 0.43 (p<0.001) and RAAD r = 0.40 (p<0.001). The association with modified Sharp score and RAAD score was maintained after adjustment for CRP, ESR, rheumatoid factor, DAS28, sex, and age in a multiple regression analysis (p = 0.018 and p = 0.04, respectively), while neither CRP nor ESR showed any independent associations. Highly significant correlations (p<0.001) were also found between calprotectin and both laboratory and clinical markers of inflammation. CONCLUSION: Calprotectin was found to significantly and independently explain the variation in the radiological and clinical assessments of joint damage. Longitudinal studies are required to examine whether calprotectin may predict the progression of joint damage in RA