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.

The calcium-binding protein S100A12 correlates with measures of disease activity in patients with rheumatoid arthritis (RA). The protein reflects neutrophil activation and the present objective was to explore in a pilot study the associations between S100A12 and other inflammatory markers, clinical assessments as well as degree of synovitis detected by a comprehensive ultrasonography (US) examination in RA patients during biologic treatment. Twenty patients with RA were examined clinically and by use of US as well as laboratory markers S100A12, calprotectin, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) before starting adalimumab, with follow-up after 1, 3, 6 and 12 months. Ultrasonographic B-mode (BM) and power Doppler (PD) assessments of 78 joints, 36 tendons/tendon groups and 2 bursas were performed, and sum US scores calculated. Wilcoxon signed rank test assessed treatment response and Spearman rank correlation test was used to calculate correlations. The concentrations of S100A12 decreased after 3 months (p < 0.01) and significant correlations were found between S100A12 and the other laboratory markers during follow-up (0.50-0.62, p < 0.05). Of the clinical assessments, S100A12 had highest correlations with the assessor’s global VAS (0.46-0.85, p < 0.05). Compared with CRP and ESR, S100A12 showed higher correlations with the sum US scores (both BM and PD), with median (range) correlation coefficients of 0.55 (0.35-0.78 (NS-p < 0.001)) for sum BM scores and 0.45 (0.27-0.75 (NS-p < 0.001)) for sum PD scores. The S100A12 protein was significantly associated with other inflammatory markers, clinical assessments as well as sum US scores, indicating that S100A12 is a potential marker of inflammation in RA patients.

ChAdOx1 nCoV-19 vaccination has been associated with the rare side effect; vaccine-induced immune thrombotic thrombocytopenia (VITT). The mechanism of thrombosis in VITT is associated with high levels of neutrophil extracellular traps (NETs). The present study examines whether key markers for NETosis, such as H3-NETs and calprotectin, as well as syndecan-1 for endotheliopathy, can be used as prognostic factors to predict the severity of complications associated with ChAdOx1 vaccination. Five patients with VITT, 10 with prolonged symptoms and cutaneous hemorrhages but without VITT, and 15 with only brief and mild symptoms after the vaccination were examined. Levels of H3-NETs and calprotectin in the vaccinated individuals were markedly increased in VITT patients compared to vaccinees with milder vaccination-associated symptoms, and a strong correlation (r ≥ 0.745, p < 0.001) was found with severity of vaccination side effects. Syndecan-1 levels were also positively correlated (r = 0.590, p < 0.001) in vaccinees to side effects after ChAdOx1 nCoV-19 vaccination. We hypothesize that the inflammatory markers NETs and calprotectin may be used as confirmatory tests in diagnosing VITT.

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.