Epitope-Specific Anti-C1q Autoantibodies in Systemic Lupus Erythematosus

ObjectiveIn patients with systemic lupus erythematosus (SLE) complement C1q is frequently targeted by autoantibodies (anti-C1q), that correlate best with active renal disease. Anti-C1q bind to largely unknown epitopes on the collagen-like region (CLR) of this highly functional molecule. Here we aimed at exploring the role of epitope-specific anti-C1q in SLE patients.MethodsFirst, 22 sera of SLE patients, healthy controls and anti-C1q positive patients without SLE were screened for anti-C1q epitopes by a PEPperMAP® microarray, expressing CLR of C1q derived peptides with one amino acid (AA) shift in different lengths and conformations. Afterwards, samples of 378 SLE patients and 100 healthy blood donors were analyzed for antibodies against the identified epitopes by peptide-based ELISA. Relationships between peptide-specific autoantibodies and SLE disease manifestations were explored by logistic regression models.ResultsThe epitope mapping showed increased IgG binding to three peptides of the C1q A- and three of the C1q B-chain. In subsequent peptide-based ELISAs, SLE sera showed significantly higher binding to two N-terminally located C1q A-chain peptides than controls (p < 0.0001), but not to the other peptides. While anti-C1q were associated with a broad spectrum of disease manifestations, some of the peptide-antibodies were associated with selected disease manifestations, and antibodies against the N-terminal C1q A-chain showed a stronger discrimination between SLE and controls than conventional anti-C1q.ConclusionIn this large explorative study anti-C1q correlate with SLE overall disease activity. In contrast, peptide-antibodies are associated with specific aspects of the disease suggesting epitope-specific effects of anti-C1q in patients with SLE.

Detection and Characterization of the Soybean Allergen Gly m 7 in Soybeans and Processed Soybean Foods

Gly m 7, a novel soybean allergen, was recently reported. In this study, we attempted to detect Gly m 7 in various soybeans and processed soybean foods using raised anti-Gly m 7 antibodies and enzyme-linked streptavidin, specifically binding to the biotin moiety of Gly m 7. There was a large difference in Gly m 7 levels in various soybean-processed foods. When Gly m 7 levels were determined, all cultivars contained this allergen almost completely, but the biotin moiety detected by streptavidin varied, suggesting that biotinylated levels of Gly m 7 might differ among cultivars. The thermal stability of Gly m 7 was determined by heating soybean extracts. During detection using anti-peptide antibodies, detectable intact Gly m 7 was gradually reduced by heating. Gly m 7 was not detected by peptide or biotin detection in worm-wounded soybeans. Soybeans were immersed in distilled water as a pretreatment step for germination, and Gly m 7 levels were compared by immersion time (4–96 h). Intact Gly m 7 was rapidly degraded in detection by both peptide and biotin moieties. This suggested that Gly m 7 was degraded by some protease(s) during germination. These results would be useful for understanding the properties or risk assessment of Gly m 7, a newly discovered soybean allergen.

A SISCAPA-based approach for detection of SARS-CoV-2 viral antigens from clinical samples

SARS-CoV-2, a novel human coronavirus, has created a global disease burden infecting > 100 million humans in just over a year. RT-PCR is currently the predominant method of diagnosing this viral infection although a variety of tests to detect viral antigens have also been developed. In this study, we adopted a SISCAPA-based enrichment approach using anti-peptide antibodies generated against peptides from the nucleocapsid protein of SARS-CoV-2. We developed a targeted workflow in which nasopharyngeal swab samples were digested followed by enrichment of viral peptides using the anti-peptide antibodies and targeted parallel reaction monitoring (PRM) analysis using a high-resolution mass spectrometer. This workflow was applied to 41 RT-PCR-confirmed clinical SARS-CoV-2 positive nasopharyngeal swab samples and 30 negative samples. The workflow employed was highly specific as none of the target peptides were detected in negative samples. Further, the detected peptides showed a positive correlation with the viral loads as measured by RT-PCR Ct values. The SISCAPA-based platform described in the current study can serve as an alternative method for SARS-CoV-2 viral detection and can also be applied for detecting other microbial pathogens directly from clinical samples.

Identification of a Novel Serological Marker in Seronegative Rheumatoid Arthritis Using the Peptide Library Approach

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation mainly affecting the joints leading to cartilage and bone destruction. The definition of seropositive or seronegative RA is based on the presence or absence of rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPAs). Other autoantibodies have been identified in the last decade such as antibodies directed against carbamylated antigens, peptidyl-arginine deiminase type 4 and v-Raf murine sarcoma viral oncogene homologue B. In order to identify relevant autoantigens, we screened a random peptide library (RPL) with pooled IgGs obtained from 50 patients with seronegative RA. Patients’ sera were then used in an ELISA test to identify the most frequently recognized peptide among those obtained by screening the RPL. Sera from age- and sex-matched healthy subjects were used as controls. We identified a specific peptide (RA-peptide) recognized by RA patients’ sera, but not by healthy subjects or by patients with other immune-mediated diseases. The majority of sera from seronegative and seropositive RA patients (73.8% and 63.6% respectively) contained IgG antibodies directed against the RA-peptide. Interestingly, this peptide shares homology with some self-antigens, such as Protein-tyrosine kinase 2 beta, B cell scaffold protein, Liprin-alfa1 and Cytotoxic T lymphocyte protein 4. Affinity purified anti-RA-peptide antibodies were able to cross react with these autoantigens. In conclusion, we identified a peptide that is recognized by seropositive and, most importantly, by seronegative RA patients’ sera, but not by healthy subjects, conferring to this epitope a high degree of specificity. This peptide shares also homology with other autoantigens which can be recognized by autoantibodies present in seronegative RA sera. These newly identified autoantibodies, although present also in a percentage of seropositive RA patients, may be considered as novel serum biomarkers for seronegative RA, which lacks the presence of RF and/or ACPAs.

Impact of Posttranslational Modification in Pathogenesis of Rheumatoid Arthritis: Focusing on Citrullination, Carbamylation, and Acetylation

Rheumatoid arthritis (RA) is caused by prolonged periodic interactions between genetic, environmental, and immunologic factors. Posttranslational modifications (PTMs) such as citrullination, carbamylation, and acetylation are correlated with the pathogenesis of RA. PTM and cell death mechanisms such as apoptosis, autophagy, NETosis, leukotoxic hypercitrullination (LTH), and necrosis are related to each other and induce autoantigenicity. Certain microbial infections, such as those caused by Porphyromonasgingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella copri, can induce autoantigens in RA. Anti-modified protein antibodies (AMPA) containing anti-citrullinated protein/peptide antibodies (ACPAs), anti-carbamylated protein (anti-CarP) antibodies, and anti-acetylated protein antibodies (AAPAs) play a role in pathogenesis as well as in prediction, diagnosis, and prognosis. Interestingly, smoking is correlated with both PTMs and AMPAs in the development of RA. However, there is lack of evidence that smoking induces the generation of AMPAs.

The Effect of Chinese Medicine Compound in the Treatment of Rheumatoid Arthritis on the Level of Rheumatoid Factor and Anti-Cyclic Citrullinated Peptide Antibodies: A Systematic Review and Meta-Analysis

Objectives: To evaluate the current evidence whether Chinese medicine compound (CMC) can reduce the serum levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP).Methods: We comprehensively searched PubMed, Embase, the Cochrane Library, China National Knowledge Infrastructure (CNKI), the Database for Chinese Technical Periodicals (VIP), and Wanfang data. We then performed a systematic review and meta-analysis of all randomized controlled trials (RCTs) assessing the CMC therapy methods. This study is registered with PROSPERO, number CRD42020216284.Results: In total, 65 studies were eligible for inclusion, including 6099 patients. The result of the meta-analysis showed that compared with common Western medicine therapy, CMC monotherapy or combined with Western medicine was able to reduce serum RF (SMD= −0.85, 95%CI −1.04 to −0.67) and anti-CCP (SMD= −0.56, 95%CI −0.79 to −0.32) levels to some extent. In the efficacy meta-analysis, a greater number of CMC-treated patients achieved the efficacy criteria after a period of treatment, where the relative risk (RR) was 1.20 [1.08, 1.33] for achieving ACR20, 1.57 [1.38, 1.78] for ACR50, and 2.21 [1.72, 2.84] for ACR70. At the same time, there was a statistically significant difference in the effective rate of the patient's TCM symptoms (RR = 1.22, 95%CI 1.19–1.26).Conclusions: Through this meta-analysis and systematic review, we found that CMC for the treatment of RA is effective in reducing RF and anti-CCP levels and might have better clinical efficacy than Western medicine monotherapy. Some active components are responsible for this efficacy and worth further exploring.