Potential Application of Anti-Cyclic Citrullinated Peptide (Anti-CCP) for the Diagnosis of Periodontal Disease in Patients with Rheumatoid Arthritis with Cut-Off Determination.

Background: Rheumatoid arthritis (RA) and periodontal disease (PD) are both characterized by an inflammatory reaction. Anti-cyclic citrullinated peptide (anti-CCP) is a common diagnostic test for RA. Anti-CCP is proposed to be used as a serological biomarker to detect PD in patients with RA.Methods: A case-control study was designed for 94 subjects; 42 patients with RA and 52 without RA. PD and its types were investigated among these subjects. An enzyme-linked immunosorbent assay (ELISA) test was used to measure anti-CCP levels in these subjects.Results: Subjects were recategorized into four groups after PD diagnosis: group 1 for RA with PD (34.04%); group 2 for RA only (10.63%); group 3 for PD only (30.85%); and group 4 for individuals without RA and PD (24.46%). Anti-CCP may be considered an effective biomarker for predicting the development of PD in RA patients based on five current results found in group 1 compared to other groups. These included the detection of significantly higher anti-CCP levels, a high sensitivity (63.15%), low specificity (50%), higher cut-off value (58.53 U/ml), high positive predictive value (94.73%), and low negative predictive value (8.69%) of anti-CCP. Chronic periodontitis is the most common and has a significant association with elevated levels of anti-CCP.Conclusion: Measuring the anti-CCP level in RA patients may be a good indicator for PD diagnosis based on the suggested cut-off value. The sensitivity of the test is sufficiently reliable to produce true positive results. Anti-CCP may also be useful in the diagnosis of PD type, especially chronic periodontitis.

In vitro elimination of autoreactive B cells from rheumatoid arthritis patients by universal chimeric antigen receptor T cells.

OBJECTIVES: Autoreactive B cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA), and B cell-depleting therapies using an antibodies, such as rituximab, have been suggested to be effective in RA treatment. However, transient B cell depletion with rituximab is associated with significant safety challenges related to global suppression of the immune system and thus increases the risks of infection and cancer development. To address selective and persistent issues associated with RA therapy, we developed a customised therapeutic strategy employing universal antifluorescein isothiocyanate (FITC) chimeric antigen receptor T cells (CAR-T cells) combined with FITC-labelled antigenic peptide epitopes to eliminate autoreactive B cell subsets recognising these antigens in RA. METHODS: For a proof-of-concept study, four citrullinated peptide epitopes derived from citrullinated autoantigens, namely, citrullinated vimentin, citrullinated type II collagen, citrullinated fibrinogen and tenascin-C, and a cyclocitrulline peptide-1 were selected as ligands for targeting autoreactive B cells; Engineered T cells expressing a fixed anti-FITC CAR were constructed and applied as a universal CAR-T cell system to specifically eliminate these protein-specific autoreactive B cells via recognition of the aforementioned FITC-labelled autoantigenic peptide epitopes. RESULTS: We demonstrated that anti-FITC CAR-T cells could be specifically redirected and kill hybridoma cells generated by immunisation with antigenic peptides, and autoreactive B cell subsets from RA patients via recognition of corresponding FITC-labelled citrullinated peptide epitopes. Additionally, the cytotoxicity of the CAR-T cells was dependent on the presence of the peptides and occurred in a dose-dependent manner. CONCLUSIONS: The approach described here provides a direction for precise, customised approaches to treat RA and can likely be applied to other systemic autoimmune diseases.

Unique autoantibody prevalence in long-term recovered SARS-CoV-2-infected individuals.

The variability in resolution of SARS-CoV-2-infections between individuals neither is comprehended, nor are the long-term immunological consequences. To assess the long-term impact of a SARS-CoV-2-infection on the immune system, we conducted a prospective study of 80 acute and former SARS-CoV-2 infected individuals and 39 unexposed donors to evaluate autoantibody responses and immune composition. Autoantibody levels against cyclic citrullinated peptide (CCP), a specific predictor for rheumatoid arthritis (RA), were significantly (p = 0.035) elevated in convalescents only, whereas both acute COVID-19 patients and long-term convalescents showed critically increased levels of anti-tissue transglutaminase (TG), a specific predictor of celiac disease (CD) (p = 0.002). Both, anti-CCP and anti-TG antibody levels were still detectable after 4-8 months post infection. Anti-TG antibodies occurred predominantly in aged patients in a context of a post-SARS-CoV-2-specific immune composition (R2 = 0.31; p = 0.044). This study shows that increased anti-CCP and anti-TG autoantibody levels can remain long-term after recovering even from mildly experienced COVID-19. The inter-relationship of the lung as viral entry side and RA- and CD-associated autoimmunity indicates that a SARS-CoV-2-infection could be a relevant environmental factor in their pathogenesis.

Do RA associated HLA-DR molecules bind citrullinated peptides or peptides from PAD4 to help the development of RA specific antibodies to citrullinated proteins?

PURPOSE: Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding a five amino acid basic motive, the shared epitope SE). Each HLA-DRB1 genotype defines a genotype specific risk of developing RA. RA is preceded by the emergence of anti citrullinated protein antibodies (ACPAs). Citrullin is a neutral version of arginin, a basic amino acid, formed after post translational modification by Peptidyl Arginyl Deiminases (PADs). HLA-DRB1 genes associated with RA are also associated with ACPAs. Two models might explain this association. Here we tested both models for prediction of HLA-DRB1 genotypic risks of developing RA. METHODS: We calculated the likelihoods for the 2 HLA-DR molecules encoded by 12 common HLA-DRB1 genotypes to bind at least one randomly chosen peptide from PAD4 or fibrinogen(native or citrullinatd) and compared them with the 12 respective HLA-DRB1genotypic risks of developing RA. RESULTS: HLA-DRB1 Genotypic risks of developing RA correlate with likelihoods of binding PAD4 peptides, not citrullinated Fibrinogen peptides. Thus, the molecular basis for the association of HLA-DR and ACPA positive RA is most likely the capability for RA associated HLA-DR molecules to bind peptides(s) from PAD4.

Antibodies to malondialdehyde-acetaldehyde modified low-density lipoprotein in patients with newly diagnosed inflammatory joint disease.

Objective:To assess antibodies to malondialdehyde-acetaldehyde-modified low-density lipoprotein (MAA-LDL) in patients with newly diagnosed inflammatory joint disease.Method: Patients with rheumatoid arthritis (RA), spondyloarthritis (SpA), and undifferentiated arthritis (UA), participating in the Northern Savo 2010 Study, were evaluated for metabolic syndrome (MetS), metabolic and inflammatory markers, antibodies to MAA-LDL, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis.Results: Among 135 newly diagnosed untreated patients, of whom 53 (39%) were diagnosed to have RA, 44 (33%) SpA, and 38 (28%) UA, 49%, 30%, and 47%, respectively, had MetS. After adjusting for age and gender, anti-MAA-LDL immunoglobulin (Ig)A (p = 0.009), IgG (p = 0.031), and IgM (p = 0.001) levels differed between the diagnostic categories, but not in patients with MetS present or absent. All antibody classes to MAA-LDL correlated with erythrocyte sedimentation rate (ESR), and IgA and IgG antibodies with high-sensitivity C-reactive protein (hs-CRP). IgA antibodies to MAA-LDL correlated with rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), fasting plasma glucose, IgA antibodies to A. actinomycetemcomitans, and in IgA and IgG antibodies to P. gingivalis.Conclusion: Among various arthritis groups, antibodies to MAA-LDL were most common in RA. Antibodies to modified lipoproteins were associated with inflammation measured by ESR and hs-CRP. IgA antibodies to MAA-LDL correlated with age, antibodies to periodontal bacteria, RF, ACPA, and fasting glucose. Associations between antibodies to MAA-LDL and antibodies to periodontal bacteria, RA-associated antibodies, inflammatory parameters, and plasma glucose already reflect cardiovascular burden in inflammatory joint diseases at diagnosis.

Fungal-induced protein hyperacetylation in maize identified by acetylome profiling.

Lysine acetylation is a key posttranslational modification that regulates diverse proteins involved in a range of biological processes. The role of histone acetylation in plant defense is well established, and it is known that pathogen effector proteins encoding acetyltransferases can directly acetylate host proteins to alter immunity. However, it is unclear whether endogenous plant enzymes can modulate protein acetylation during an immune response. Here, we investigate how the effector molecule HC-toxin (HCT), a histone deacetylase inhibitor produced by the fungal pathogen Cochliobolus carbonum race 1, promotes virulence in maize through altering protein acetylation. Using mass spectrometry, we globally quantified the abundance of 3,636 proteins and the levels of acetylation at 2,791 sites in maize plants treated with HCT as well as HCT-deficient or HCT-producing strains of C. carbonum Analyses of these data demonstrate that acetylation is a widespread posttranslational modification impacting proteins encoded by many intensively studied maize genes. Furthermore, the application of exogenous HCT enabled us to show that the activity of plant-encoded enzymes (histone deacetylases) can be modulated to alter acetylation of nonhistone proteins during an immune response. Collectively, these results provide a resource for further mechanistic studies examining the regulation of protein function by reversible acetylation and offer insight into the complex immune response triggered by virulent C. carbonum.

Prevalence of anti-cyclic citrullinated peptide antibodies in patients with spondyloarthritis: A retrospective study.

OBJECTIVES: Anti-cyclic citrullinated peptide (CCP) antibodies are frequently detected in the sera of patients with rheumatoid arthritis (RA). However, recent studies have revealed a potentially high prevalence rate of these antibodies in patients with other rheumatic disorders, causing confusion while diagnosing RA. Therefore, this study aimed to evaluate the positive rate of anti-CCP antibodies in other chronic arthritis diseases focusing on patients with spondyloarthritis (SpA). METHODS: A total of 109 patients who were diagnosed with SpA at Yukioka Hospital from 1993 to 2018 were included in this retrospective analysis, including patients with ankylosing spondylitis (AS); psoriatic arthritis (PsA); synovitis, acne, pustulosis, hyperostosis, and osteitis syndrome (SAPHO); undifferentiated spondyloarthritis (uSpA); reactive arthritis (ReA); and inflammatory bowel disease-associated SpA (IBD). RESULTS: Overall, 15.3% (16/109) of patients with SpA were positive for anti-CCP antibodies, including 2.3% (1/43) in AS, 23.1% (3/13) in SAPHO, 35.0% (7/20) in PsA, 14.8% (4/27) in uSpA, 0% (0/3) in ReA, and 33.3% (1/3) in IBD. CONCLUSION: PsA patients have a significantly higher prevalence rate of positive anti-CCP antibodies among SpA patients, and the positive rates in SAPHO and uSpA were also high. These findings provide insight into the heterogeneity of SpA with relevance for RA differential diagnosis.

Preclinical rheumatoid arthritis and rheumatoid arthritis prevention.

PURPOSE OF REVIEW: This review is to provide an update on the current understanding of rheumatoid arthritis (RA) development related to disease development prior to the onset clinically apparent synovitis (i.e. Pre-RA), and opportunities for disease prevention. RECENT FINDINGS: A growing number of studies have demonstrated that serum elevations of autoantibodies rheumatoid factor, antibodies to citrullinated protein/peptide antigens (ACPAs) and antibodies to other posttranslationally modified proteins (e.g. carbamylated proteins) are highly predictive of future development of inflammatory arthritis/RA during a period that can be termed Pre-RA. Other factors including genetic, environmental, symptoms and imaging findings can also enhance prediction. Moreover, several novel biomarkers and changes in autoantibodies (e.g. glycosylation of variable domains) have been identified in Pre-RA. There has also been growing evidence that initiation and propagation of RA-related autoimmunity during the Pre-RA phase may be related to mucosal processes. The discovery of Pre-RA has also underpinned the development of several clinical prevention trials in RA; specifically, the PRAIRI study demonstrated that a single dose of rituximab can delay the onset of clinically apparent IA in at-risk individuals. Additional studies are evaluating the ability of drugs including abatacept, hydroxychloroquine and methotrexate to prevent or delay future RA. SUMMARY: The results from ongoing natural history and prevention trials in RA should further inform several critical issues in RA prevention including identification and enrolment of individuals at high-risk of imminent RA, the efficacy, safety and cost-effectiveness of prevention, and potentially the identification of new targets for prevention.

Peptidoglycan-Associated Cyclic Lipopeptide Disrupts Viral Infectivity.

Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from Bacillus subtilis reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection in vivo Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection.IMPORTANCE In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.

Secretory antibodies to citrullinated peptides in plasma and saliva from rheumatoid arthritis patients and their unaffected first-degree relatives.

The aim of this study was to evaluate secretory antibodies to citrullinated proteins (ACPA) in plasma and immunoglobulin (Ig)A ACPA in saliva from patients with rheumatoid arthritis (RA) and their unaffected first-degree relatives (FDRs). Patients with RA (n = 194) and first-degree relatives unaffected by RA (n = 191) were recruited for analysis of secretory antibodies to second-generation cyclic citrullinated peptides (anti-CCP) in plasma. From a subpopulation (25 RA patients, 21 first-degree relatives and 11 controls), saliva samples were obtained for IgA anti-CCP analysis. The presence of secretory ACPA was compared between subject categories, and related to genetic and environmental risk factors. Secretory ACPA occurred in 37 (19%) plasma samples from patients with RA, but only in two (1%) of FDRs. IgA ACPA in saliva was found in three of 25 (12%) patients with RA, but not in any of the 21 FDRs (< 5%). No significant associations were seen between the presence of secretory ACPA and SE or smoking, either among RA patients or among FDRs. Despite occurring in 19% of RA plasma, secretory ACPA was rare in both saliva and plasma among FDRs, even among those positive for conventional ACPA of non-mucosal origin. Longitudinal studies are warranted to determine whether circulating secretory ACPA occurs before or in parallel with the development of clinical arthritis.

Anti-carbamylated proteins antibody repertoire in rheumatoid arthritis: evidence of a new autoantibody linked to interstitial lung disease.

OBJECTIVE: To analyse the association between anti-carbamylated protein antibodies (Anti-CarP) and interstitial lung disease (ILD) in rheumatoid arthritis (RA) patients. METHODS: Cross-sectional study including RA patients fulfilling the 2010 ACR/EULAR criteria. The main population comprised two groups: (1) RA patients diagnosed with RA-ILD (RA-ILD group); (2) RA patients without ILD (non-ILD RA group). Non-ILD RA patients in whom ILD was suspected underwent a diagnostic work-up and, if ILD was diagnosed, were switched to the RA-ILD group. ILD was diagnosed by high-resolution computed tomography and confirmed by a multidisciplinary committee. An independent replication sample was also obtained. Three Anti-CarP IgG autoantibodies against fetal calf serum (Anti-FCS), fibrinogen (Anti-Fib) and chimeric fibrine/filagrine homocitrullinated peptide (Anti-CFFHP) and one Anti-CarP IgA against FCS (Anti-FCS-IgA) were determined by home-made ELISA. Associations between Anti-CarP and ILD were analysed using multivariable logistic regression adjusted by smoking, sex, age, RA disease duration, rheumatoid factor and anticitrullinated protein antibodies. RESULTS: We enrolled 179 patients: 37 (21%) were finally diagnosed with RA-ILD. Anti-CarP specificities were more frequent in RA-ILD patients (Anti-FCS 70% vs 43%; Anti-Fib 73% vs 51%; Anti-CFFHP 38% vs 19%; Anti-CarP-IgA 51% vs 20%, p<0.05 for all comparisons). Serum titers of Anti-CarP were significantly higher in RA-ILD patients. Anti-CarP specificities showed a robust effect towards increasing the odds of ILD in the multivariate analysis (Anti-FCS (OR: 3.42; 95% CI: 1.13 to 10.40), Anti-Fib (OR: 2.85; 95% CI: 0.83 to 9.70), Anti-CFFHP (OR: 3.11; 95% CI: 1.06 to 9.14) and Anti-FCS-IgA (OR: 4.30; 95% CI: 1.41 to 13.04)). Similar findings were observed in the replication sample. CONCLUSIONS: Anti-CarP were strongly associated with ILD. The role of homocitrullination in RA-ILD merits further investigation.

Factors Influencing the Immunogenicity and Immunotoxicity of Cyclic RGD Peptide-Modified Nanodrug Delivery Systems.

c(RGDyK)-modified liposomes have been shown to be immunogenic and potentially trigger acute systemic anaphylaxis upon repeated intravenous injection in both BALB/c nude mice and ICR mice. However, questions concerning the potential influence of mouse strains, immunization routes, drug carrier properties, and changes in c(RGDyK) itself on the immunogenicity and resultant immunotoxicity (anaphylaxis) of cyclic RGD peptide-modified nanodrug delivery systems remain unanswered. Here, these potential impact factors were investigated, aiming to better understand the immunological properties of cyclic RGD peptide-based nanodrug delivery systems and seek for solutions for this immunogenicity-associated issue. It was revealed that anaphylaxis caused by intravenous c(RGDyK)-modified drug delivery systems might be avoided by altering the preimmunization route (i.e., subcutaneous injection), introducing positively charged lipids into the liposomes and by using micelles or red blood cell membrane (RBC)-based drug delivery systems as the carrier. Different murine models showed different incidences of anaphylaxis following intravenous c(RGDyK)-liposome stimulation: anaphylaxis was not observed in both SD rats and BALB/c mice and was less frequent in C57BL/6 mice than that in ICR mice. In addition, enlarging the peptide ring of c(RGDyK) by introducing amino sequence serine-glycine-serine reduced the incidence of anaphylaxis post the repeated intravenous c(RGDyKSGS)-liposome stimulation. However, immunogenicity of cyclic RGD-modified drug carriers could not be reversed, although some reduction in IgG antibody production was observed when ICR mice were intravenously stimulated with c(RGDyK)-modified micelles, RBC membrane-based drug delivery systems and c(RGDyKSGS)-liposomes instead of c(RGDyK)-liposomes. This study provides a valuable reference for future application of cyclic RGD peptide-modified drug delivery systems.

Antibodies against carbamylated vimentin exist in systemic lupus erythematosus and correlate with disease activity.

OBJECTIVES: Antibodies against carbamylated protein (anti-CarP) were found to be a promising marker to evaluate joint damage and disease activity in patients with rheumatoid arthritis (RA). However, whether anti-CarP antibodies were present in systemic lupus erythematosus (SLE) remained ambiguous. We have therefore undertaken this study to assess the levels of serum anti-CarP antibodies and to evaluate their clinical value in SLE. METHODS: Serum levels of antibodies against carbamylated-vimentin (anti-Carp) were measured by enzyme immunosorbent assay in 100 patients with SLE, 76 with RA, 17 with primary Sjögren syndrome (pSS), and 68 healthy controls. Data analyses between anti-Carp antibodies and other laboratory measures were performed using SPSS 24 software for Windows. RESULTS: The levels of serum anti-CarP antibodies in patients with SLE were significantly higher than those in healthy controls. In addition, anti-CarP antibodies were present in SLE patients lacking the disease-specific antibodies, including anti-Smith-negative patients (24.4%, 21/86), anti-dsDNA-negative patients (29.3%, 12/41), anti-nucleosome-negative patients (21.4%, 9/42), and antiribosomal P protein antibody-negative patients (23.7%, 18/76). There were significant differences between the anti-CarP-positive and anti-CarP-negative SLE patients in clinical and laboratory features, such as age, erythrocyte sedimentation rate (ESR), C-reactive protein, rheumatoid factor, third-generation cyclic citrullinated peptide (CCP3), anticardiolipin, D-dipolymer, complement 3, immunoglobulin G (IgG), red blood cell count (RBC) and hemoglobin. After adjusting for age and disease duration, the high levels of anti-CarP antibodies were still correlated with low RBC, hemoglobin and high ESR, IgG and CCP3. Active SLE patients demonstrated higher anti-CarP IgG than inactive patients. Moreover, the levels of anti-CarP were significantly higher in SLE patients with arthralgia and/or arthritis than in those without joint involvement. CONCLUSIONS: Anti-CarP antibodies were present in SLE patients and associated with the disease severity. These might provide a potential supplement to other specific autoantibodies for diagnosis of SLE and serve as a promising marker for measuring joint damage in the disease.

Increased serum levels of microfibrillar-associated protein 4 (MFAP4) are not associated with clinical synovitis in rheumatoid arthritis but may reflect underlying cardiovascular comorbidity.

OBJECTIVES: To study circulating MFAP4 in rheumatoid arthritis (RA) and its associations with clinical phenotype. METHODS: Early RA (ERA): 47 patients with newly diagnosed, treatment naïve RA were included. Serum MFAP4, clinical and laboratory disease variables were recorded serially during 12 months of intensive synovitis suppressive treatment. Long-standing RA (LRA): 317 patients participated, all receiving DMARD treatment. Disease activity, autoantibody status, extra-articular manifestations and cardiovascular morbidity were recorded. Paired serum and synovial fluid samples were obtained from 13 untreated ERA patients. Healthy blood donors served as reference points. MFAP4 was quantified by AlphaLISA immunoassay. Univariate, multivariate and mixed effects regression models were applied in the statistical analysis. RESULTS: ERA: MFAP4 increased from baseline and was significantly elevated at the 12-month follow-up, 17.8 U/l [12.6;24.1] vs. healthy controls, 12.7 U/l [9.5;15.6], p<0.001. MFAP4 did not correlate with joint counts or C-reactive protein. LRA: MFAP4 was increased, 25.9 U/l [20.4;33.7] vs. healthy controls, 17.6 U/l [13.7;21.2], p<0.0001, but did not correlate with disease activity measures or presence of extra-articular manifestations. Notably, MFAP4 correlated inversely with smoking (p<0.0001) and presence of antibodies against cyclic citrullinated peptides (anti-CCP), p=0.005. There was a positive association with systolic blood pressure, p=0.001 and co-occurrence of three cardiovascular events and/or risk factors, p<0.0001. The serum:synovial fluid MFAP4 ratio was 2:1. CONCLUSIONS: MFAP4 increases from diagnostic baseline despite intensive treatment but does not associate with synovitis at early or late stages of RA. Correlation patterns indicate that increased MFAP4 may reflect enhanced RA-related vascular remodelling.

Is periodontitis a prognostic factor in order to indicate antibodies against citrullinated peptides in patients with rheumatoid arthritis?

OBJECTIVES: In this cross-sectional study we investigated antibody titres against cyclic citrullinated peptides derived from filaggrin (anti-CCP) and citrullinated α-enolase (anti-CEP-1) among patients with RA as a function of periodontal findings. METHODS: 107 patients with RA (median age 56 years, 75% females) were included. For periodontal diagnoses missing teeth, periodontal epithelial surface area, periodontal inflamed surface area and periodontal diagnosis according to the working group's guidelines of the Center for Disease Control and Prevention were determined. Subgingival bacterial DNA of five periodontopathic bacteria was assessed by PCR with sequence-specific oligonucleotides. Anti-CCP and anti-CEP-1 antibodies in plasma samples were investigated using enzyme-linked immunosorbent assays. Low resolution human leukocyte antigen (HLA) typing was carried out using PCR with sequence-specific primers. RESULTS: PESA was found associated with a low adjusted odds ratio for anti-CCP positivity (OR=1.002, p=0.040). All patients who were infected with Aggregatibacter actinomycetemcomitans were simultaneously anti-CCP positive (p=0.043). HLA-DRB1*13 lowered the adjusted odds ratio for anti-CCP (OR=0.073, p=0.002) and anti-CEP-1 (OR=0.068, p=0.018) positivity whereas HLA-DRB1*07 indicated a lower risk only for demonstrable anti-CCP antibodies (OR=0.079, p=0.004). HLA-DRB1*04 was associated with increased adjusted odds ratio for anti-CEP-1 positivity (OR=4.154, p=0.005) and the simultaneous proof of both investigated autoantibodies (OR=3.725, p=0.011). CONCLUSIONS: Among patients with RA periodontitis may be a minor risk factor for anti-CCP positivity. Our data first provide evidence that an infection with A. actinomycetemcomitans is associated with an increased formation of anti-CCP. HLA phenotype proved to be a significant risk indicator for both investigated antibodies.

Cutting Edge: B Cells Expressing Cyclic Citrullinated Peptide-Specific Antigen Receptor Are Tolerized in Normal Conditions.

Generation of neoantigens by citrullination is implicated in the production of anti-citrullinated protein Abs in rheumatoid arthritis, but citrullination is also a physiological process. To verify whether citrullin-specific B cells are immunologically ignorant or tolerant in normal conditions, transgenic (Tg) mice expressing IgM with the V region of an anti-cyclic citrullinated peptide (CCP) mAb cloned from a rheumatoid arthritis patient were generated. CCP-specific B cells developed in the anti-CCP IgM Tg mice with an alteration of bone marrow B cell fractions, and the number of mature B cells decreased compared with wild-type or the control anti-influenza nucleoprotein-specific IgM Tg mice. In addition, B cells in anti-CCP IgM Tg mice are functionally anergic. Thus, tolerance is induced in CCP-specific B cells in vivo, suggesting that the immune systems are naturally exposed to citrullinated Ags, and anti-CCP Ab production requires additional steps beyond the generation of neoantigens by citrullination.

Cyclic Peptide Mimotopes for the Detection of Serum Anti-ATIC Autoantibody Biomarker in Hepato-Cellular Carcinoma.

Tumor-associated (TA) autoantibodies have been identified at the early tumor stage before developing clinical symptoms, which holds hope for early cancer diagnosis. We identified a TA autoantibody from HBx-transgenic (HBx-tg) hepatocellular carcinoma (HCC) model mouse, characterized its target antigen, and examined its relationship to human HCC. The mimotopes corresponding to the antigenic epitope of TA autoantibody were screened from a random cyclic peptide library and used for the detection of serum TA autoantibody. The target antigen of the TA autoantibody was identified as an oncogenic bi-functional purine biosynthesis protein, ATIC. It was upregulated in liver cancer tissues of HBx-tg mouse as well as human HCC tissues. Over-expressed ATIC was also secreted extracellularly via the cancer-derived exosomes, which might cause auto-immune responses. The cyclic peptide mimotope with a high affinity to anti-ATIC autoantibody, CLPSWFHRC, distinguishes between serum samples from HCC patients and healthy subjects with 70.83% sensitivity, 90.68% specificity (AUC = 0.87). However, the recombinant human ATIC protein showed a low affinity to anti-ATIC autoantibody, which may be incompatible as a capture antigen for serum TA autoantibody. This study indicates that anti-ATIC autoantibody can be a potential HCC-associated serum biomarker and suggests that autoantibody biomarker's efficiency can be improved by using antigenic mimicry to native antigens present in vivo.

Subcalcaneal bursitis as the initial manifestation of rheumatoid arthritis: ultrasonographic observation of two cases.

In early rheumatoid arthritis (RA), proliferative synovitis sometimes occurs earlier in the tenosynovium or bursal synovium than in the articular synovium. Here we report two patients who presented with subcalcaneal bursitis while progressing from undifferentiated arthritis with high-titer anti-CCP antibodies (ACPA) to a diagnosis of RA. They had initially presented with palindromic transient pain in the hands and the feet. They were strongly positive for ACPA and negative for rheumatoid factor (RF) at the onset of symptoms. A few years later, they developed persistent plantar heel pain and underwent musculoskeletal ultrasonography (MSUS). MSUS revealed subcalcaneal bursitis with synovial proliferation. At that time, they became positive for RF and they were clinically diagnosed and began receiving treatment for RA. They developed overt synovitis in their wrists and fingers several months later. To the best of our knowledge, this is the first report on MSUS-detection of subcalcaneal bursitis with synovial proliferation in patients in the very early phase of RA, although there have been many reports of forefoot bursitis. These cases suggest that MSUS scanning of the plantar surface of the heel may be useful for patients with plantar heel pain who are suspected of having a very early phase of RA, because proliferative synovitis can be detected as subcalcaneal bursitis.

Predicting factors for disappearance of anti-mutated citrullinated vimentin antibodies in sera of patients with rheumatoid arthritis.

Objectives: We aimed to determine the predicting factors for disappearance of anti-mutated citrullinated vimentin antibody (anti-MCV Ab) in sera from rheumatoid arthritis (RA) patients.Methods: In 2013, 95 RA patients whose Disease Activity Score with erythrocyte sedimentation rate were moderate to severe (DAS28-ESR ≥3.2) at baseline were enrolled. Titers of anti-MCV Ab and anti-cyclic citrullinated peptide (anti-CCP) Ab for 2013 and 2014 were measured. The association of anti-MCV disappearance with disease activity, treatment, interstitial lung disease (ILD), and serum markers of ILD were retrospectively examined. Predicting factors of anti-MCV disappearance were determined by multivariable analysis.Results: While anti-CCP positivity rate did not change during the year, anti-MCV Ab changed from positive to negative in 18 patients (=19.0%). Continuous biological disease-modifying anti-rheumatic drug use, prednisolone dose (≥5.0 mg daily), and low KL-6 level (<191 U/mL) were determined as predicting factors of anti-MCV disappearance by multivariable analysis. In our cohort, anti-MCV Ab disappearance was not linked to clinical and radiological improvement.Conclusion: Different from anti-CCP Ab, anti-MCV Ab in sera from RA patients can disappear in a year. Some predicting factors for such negative seroconversion were found, whereas clinical significance of anti-MCV Ab disappearance was undetermined.

Valine 11 and phenylalanine 13 have a greater impact on the T-cell response to citrullinated peptides than the 70-74 shared epitope of the DRB1 molecule in macaques.

OBJECTIVES: Various rheumatoid arthritis (RA) HLA-DRB-1 risk haplotypes have been regrouped under the shared epitope (SE) in position 70-74. The presence of Valine in position 11 (Val11) and phenylalanine in position 13 (Phe13) are also associated with RA, but it is impossible to differentiate their role compared with the SE since they are in strong linkage disequilibrium (LD) in humans. Similar to humans, certain macaques express the SE (H6). We analysed the effect of various DRB1 haplotypes on T-cell response to citrullinated peptides (Cit-P) in macaques. METHODS: Six H6 and six non-H6 macaques were immunized with four Cit-P. T-cell response was assessed using Interferon γ enzyme-linked immunospot. RESULTS: Animals developed a specific anti-Cit-P T-cell response. Surprisingly, H6 animals had a significantly lower T-cell response than non-H6. In macaques, the 70-74 SE and the Val11 are on separate haplotypes. Presence of Val11 was strongly associated with the anti-Cit-P T-cell response, whatever the 70-74 sequence was. This response was amplified in case of presence of Phe13. CONCLUSION: The absence of LD between Val11 and SE in macaques allowed us to demonstrate that the most important HLA positions to induce a T-cell response against Cit-P were Val11 and Phe13 and not the 70-74 SE.