Type 1 Diabetes as a Model for Prediction and Diagnosis

Objectives: Determine the frequency, clinical phenotype, and prognostic implications of antibodies against cyclic citrullinated peptides in patients with type 1 autoimmune hepatitis.

Methods: Three hundred and ninety-five serum samples from 179 patients were tested by enzyme-linked immunosorbent assay, and findings correlated with clinical and histological features, frequency of HLA DR3 and DR4, and treatment outcome.

Results: Twenty patients (11%) had antibodies against cyclic citrullinated peptides. Seropositivity was associated with a higher frequency of rheumatoid arthritis (RA) (25 vs. 0%, P < 0.001). Patients with antibodies against cyclic citrullinated peptides also had a significantly greater occurrence of histological cirrhosis at presentation (47 vs. 20%, P = 0.01) and death from hepatic failure than seronegative patients (25 vs. 9%, P = 0.04). Cirrhosis at presentation occurred more commonly in the patients with antibodies against cyclic citrullinated peptides and RA than in the other patients (100 vs. 21%, P = 0.005).

Conclusions: Antibodies against cyclic citrullinated peptides occur in a subgroup of patients with type 1 autoimmune hepatitis who have a greater occurrence of cirrhosis at presentation and death from hepatic failure. Their presence with RA at accession characterizes a subgroup with cirrhosis.     

HLA and RA revisited: citrullinated food for the SE hypothesis, the DR6 effect, and NIMA

An obvious way to unravel the apparently complex association between human leukocyte antigen (HLA) and rheumatoid arthritis (RA) is to reduce the heterogeneity of this multifactorial disease. Recently we have discovered that shared epitope (SE)-positive HLA-DRB1 alleles are exclusively associated with a subgroup of RA patients that test positive for auto-antibodies against cyclic citrullinated peptides. Further studies suggested that SE-positive alleles are classical immune response genes for the development of these antibodies. On the basis of these and other data we formulated a two-hit model for the pathogenesis of RA which incorporates a novel "citrullinated" SE hypothesis. About 5 years ago Zanelli et al. reported that HLA-DR6 (*1301 and *1302) and some other DR alleles that share the DERAA-sequence on amino acids 70-74 of their third hypervariable region are associated with protection from (severe) RA. Recently we corroborated these data in a large prospective cohort study and demonstrated that protection was observed both in the presence and in the absence of a SE susceptibility allele on the other haplotype. Finally we review the state of the art of the association of noninherited maternal HLA antigens with both susceptibility to and protection from RA.     

Frequency and significance of antibodies to soluble liver antigen/liver pancreas in variant autoimmune hepatitis

BACKGROUND: Antibodies to soluble liver antigen/liver pancreas are highly specific markers of autoimmune hepatitis. AIMS: Determine the frequency and clinical significance of these antibodies in the variant syndromes. METHODS: Antibodies to soluble liver antigen/liver pancreas were determined in 28 patients with variant forms, including 10 with cryptogenic chronic hepatitis and 18 with cholestatic variants. One hundred and seventy-two patients with classical autoimmune hepatitis were similarly tested. RESULTS: Seven of the 28 patients with variant forms had the antibodies, and this frequency was not statistically different than that in classical disease (25 vs. 12%, p = 0.08). Antibodies were most common in patients with cryptogenic chronic hepatitis (40%). Seropositive patients were indistinguishable from seronegative patients with variant forms, and they responded as well to corticosteroid therapy as patients with autoimmune hepatitis. Relapse after corticosteroid withdrawal invariably occurred in the seropositive patients whether with variant or classical disease, and HLA DR3 was more common in the seropositive patients with variant forms than in normal subjects (60 vs. 15%, p = 0.03). CONCLUSIONS: Antibodies to soluble liver antigen/liver pancreas occur commonly in the variant forms of autoimmune hepatitis and identify patients that closely resemble classical disease. Seropositivity is associated with relapse after corticosteroid withdrawal and HLA DR3. The antibodies may be surrogate markers of a genetic propensity to relapse that is independent of clinical phenotype.     

Association between shared epitope and anti-citrullinated peptide antibodies in rheumatoid arthritis: A systematic review and meta-analysis

The group of major histocompatibility complex (MHC) alleles known as shared epitope (SE) is to date the strongest rheumatoid arthritis (RA) genetic risk factor. Many studies have shown that the measurement of anti-citrullinated peptides antibodies would be useful in the diagnosis and follow-up of RA.Our aim is to determine the magnitude of the association between the possession of SE alleles and serum positive titres of antibodies against citrullinated peptides.Our selection criteria included case-control or cohort studies, where data involving antibodies against citrullinated peptides and SE in RA patients were available. No date or language restrictions were imposed.Bibliographical databases MEDLINE, Cochrane Database of Systematic Reviews and EMBASE were searched for pertinent literature. Two reviewers independently identified relevant citations and extracted data. Data extraction was then checked by two different reviewers.Five published and one unpublished (own data) studies were included in the final meta-analysis. Overall, 2700 European descent RA patients were included in this meta-analysis. A significant association between SE and positive titres of serum antibodies against citrullinated peptides [OR(95% CI) = 3.19 (2.21-4.60)] was found.Positive titres for antibodies against citrullinated peptides are threefold more frequent in RA patients who carry SE alleles than in those patients lacking them.     

Anti‐citrullinated glucose‐6‐phosphate isomerase peptide antibodies in patients with rheumatoid arthritis are associated with HLA‐DRB1 shared epitope alleles and disease activity

To identify and characterize anti‐citrullinated glucose‐6‐phosphate isomerase (GPI) peptide antibodies in patients with rheumatoid arthritis (RA). Nine GPI arginine‐bearing peptides in human GPI protein were selected and cyclic citrullinated GPI peptides (CCG‐1–9) were constructed. Samples were obtained from RA (n = 208), systemic lupus erythematosus (SLE) (n = 101), Sjögren's syndrome (SS; n = 101) and healthy controls (n = 174). Antibodies against CCG‐1–9 were measured, and anti‐citrullinated α‐enolase‐1 (CEP‐1), ‐cyclic citrullinated peptides (CCP) and ‐GPI proteins antibodies were also examined. Patients with RA were genotyped for HLA‐DRB1. The numbers of shared epitope (SE) alleles were counted and compared with those of the autoantibodies. Rabbit GPI was citrullinated with rabbit peptidylarginine deiminase and immunoblot analysis of RA sera performed. The levels of autoantibodies were compared before and after treatment with TNF antagonists in 58 RA patients. Anti‐CCG‐2, ‐4 and ‐7 antibodies were detected in 25·5, 33·2 and 37·0% patients with RA, respectively, and these antibodies were very specific for RA (specificity, 98·1–99·7%). Altogether, 44·2, 86·1 and 13·9% of RA sera were positive for anti‐CEP‐1, ‐CCP and ‐GPI protein antibodies, respectively. Anti‐CCG‐2, ‐4 and ‐7 antibodies were correlated with anti‐CCP and anti‐CEP‐1 antibodies and with the presence of HLA‐DRB1 SE alleles. Citrullinated GPI protein was detected using RA sera. Treatment with tumour necrosis factor antagonists reduced significantly the levels of anti‐CCG‐2 and ‐7 but not of anti‐CEP‐1 antibodies. This is the first report documenting the presence of anti‐CCG antibodies in RA. Anti‐CCG‐2 and ‐7 antibodies could be considered as markers for the diagnosis of RA and its disease activity.     

Frequency and Significance of Antibodies to Soluble Liver Antigen/Liver Pancreas in Variant Autoimmune Hepatitis

Background: Antibodies to soluble liver antigen/liver pancreas are highly specific markers of autoimmune hepatitis. Aims: Determine the frequency and clinical significance of these antibodies in the variant syndromes. Methods: Antibodies to soluble liver antigen/liver pancreas were determined in 28 patients with variant forms, including 10 with cryptogenic chronic hepatitis and 18 with cholestatic variants. One hundred and seventy-two patients with classical autoimmune hepatitis were similarly tested. Results: Seven of the 28 patients with variant forms had the antibodies, and this frequency was not statistically different than that in classical disease (25 vs. 12%, p = 0.08). Antibodies were most common in patients with cryptogenic chronic hepatitis (40%). Seropositive patients were indistinguishable from seronegative patients with variant forms, and they responded as well to corticosteroid therapy as patients with autoimmune hepatitis. Relapse after corticosteroid withdrawal invariably occurred in the seropositive patients whether with variant or classical disease, and HLA DR3 was more common in the seropositive patients with variant forms than in normal subjects (60 vs. 15%, p = 0.03). Conclusions: Antibodies to soluble liver antigen/liver pancreas occur commonly in the variant forms of autoimmune hepatitis and identify patients that closely resemble classical disease. Seropositivity is associated with relapse after corticosteroid withdrawal and HLA DR3. The antibodies may be surrogate markers of a genetic propensity to relapse that is independent of clinical phenotype.     

A transmembrane polymorphism in FcgammaRIIb (FCGR2B) is associated with the production of anti-cyclic citrullinated peptide autoantibodies in Taiwanese RA

The aim of the current study was to determine whether the FcgammaRIIb 187-Ile/Thr polymorphism is a predisposition factor for subtypes of RA defined by disease severity and production of autoantibodies against cyclic citrullinated peptides (anti-CCPs) in Taiwanese RA patients. Genotype distributions and allele frequencies of FcgammaRIIb 187-Ile/Thr were compared between 562 normal healthy controls and 640 RA patients as stratified by clinical parameters and autoantibodies. Significant enrichment of 187-Ile allele was observed in RA patients positive for anti-CCP antibodies as compared with the anti-CCP negative RA patients (P=0.001, OR 1.652 (95% CI 1.210-2.257)) or as compared with the normal controls (P=0.005, OR 1.348 (95% CI 1.092-1.664)). In addition, 187-Ile allele was found to be enriched in RA patients positive for rheumatoid factor (RF) compared to the RF negative RA patients (P=0.024, OR 1.562 (95% CI 1.059-2.303)). Furthermore, the homozygotes were enriched in destructive male RA patients (P=0.035; OR 2.038 (95% CI 1.046-3.973)) and the 187-Ile allele was associated with early-onset of RA in Taiwanese patients (P=0.045, OR 1.548 (95% CI 1.007-2.379)). Thus, FcgammaRIIb SNP 187-Ile/Thr may influence the RA phenotypes in Taiwanese RA.     

Subgroups of Sjögren syndrome patients according to serological profiles

Sj?gren Syndrome (SS) is a systemic, autoimmune disorder characterized by lymphocytic infiltration of the exocrine glands. Different clinical associations have been described for each of the diverse autoantibodies found in SS patients. Antibodies directed against the Ro/La ribonucleoprotein complexes have been correlated with younger age, more severe dysfunction of the exocrine glands and a higher prevalence of extraglandular manifestations. Anti-nuclear antibodies and rheumatoid factors have been associated to extraglandular manifestations and an active immunological profile, while cryoglobulins are markers of more severe disease and correlate to lymphoma development and death. Antibodies to cyclic citrullinated peptides are scarce in SS and have been linked in some cases to the development of non-erosive arthritis. Furthermore, the presence of anti-mitochondrial antibodies and anti-smooth muscle antibodies in the sera of primary SS patients is considered indicative of primary biliary cirrhosis and autoimmune hepatitis, respectively. In addition, anti-centromere antibodies have been associated with a clinical phenotype intermediate between primary SS and systemic sclerosis, while antibodies against carbonic anhydrase have been related to renal tubular acidosis. Finally, an association of anti-muscarinic antibodies with cytopenias and a higher disease activity has also been described in primary SS. In conclusion, although not all of the above mentioned antibodies are useful for predicting distinct patient subgroups in SS, knowledge of the clinical associations of the different autoantibody specificities encountered in SS can advance our understanding of the disease and improve patient management.     

Prevalence and significance of antibodies to citrullinated human papilloma virus-47 E2345-362 in rheumatoid arthritis

It has been confirmed that antibodies to citrullinated profilaggrin(306-324) may play important roles in RA. In this study, human papilloma virus (HPV)-47 E2(345-362), homologous to profilaggrin(306-324), was found using the NCBI BLAST program. Then, E2(345-362) and citrullinated E2(345-362), with arginine(348) replaced by citrulline, were synthesized. The presence of antibodies against these peptides was examined by an enzyme-linked immunosorbent assay. Associations between these antibodies and the clinical and laboratory features of RA were evaluated. Although the prevalence and AU value of antibodies to the E2(345-362) peptide were similar in RA and other rheumatic diseases, those of antibodies to the citrullinated E2(345-362) peptide were significantly higher in RA than in other rheumatic diseases. Additionally, sera that were preincubated with cyclic citrullinated peptide (CCP) demonstrated lower AU values of anti-citrullinated E2(345-362) peptide antibodies. Moreover, the prevalence of anti-CCP antibodies and that of anti-peptidylarginine deiminase (PADI4) antibodies in anti-citrullinated E2(345-362)-positive patients were all higher than those of anti-citrullinated E2(345-362)-negative patients. There were significant correlations between anti-citrullinated E2(345-362) and anti-PADI4. RA patients with antibodies to citrullinated E2(345-362) had higher DAS28 scores, erythrocyte sedimentation rates, and radiographic progression than those without the antibodies. These results suggest that HPV-47 E2 may act as an autoantigen in RA. The increase in PADI4 may make it easier to citrullinate the HPV-47 E2(345-362) peptide, leading to the subsequent immune responses.     

Antilactoferrin antibodies in autoimmune liver disease

Antilactoferrin antibodies have been reported in patients with several autoimmune disorders, including primary biliary cirrhosis, autoimmune hepatitis and autoimmune cholangitis. We investigated the prevalence and the clinical significance of such autoreactivity in patients with autoimmune and viral chronic liver disease. Sera from 39 patients with autoimmune hepatitis, 51 with primary biliary cirrhosis, 17 with autoimmune cholangitis, 24 with primary sclerosing cholangitis and 28 with HCV-related chronic hepatitis were studied. Positivity for antilactoferrin antibodies was evaluated by Western immunoblotting with purified human lactoferrin. Antilactoferrin antibodies were detected more often in autoimmune liver disorders (25% autoimmune hepatitis, 25% primary biliary cirrhosis, 35% autoimmune cholangitis, 29% primary sclerosing cholangitis) than in HCV-related chronic hepatitis (3.5%, P < 0.02 versus all). Positivity for antilactoferrin antibodies was not associated with a particular clinical or biochemical profile of the underlying liver disease. No correlation was observed between antilactoferrin reactivity and perinuclear antineutrophil cytoplasmic antibodies. Antilactoferrin antibodies are present significantly more often in autoimmune than in viral liver disorders, but they cannot be considered the serological marker of a specific autoimmune liver disease.     

Recognition of new citrulline‐containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients

Anti‐citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline‐ and arginine‐containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline‐peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline‐peptides identify antibody‐secreting cells in in vitro cultures of RA B cells. Recognition of citrulline‐ and arginine‐containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide‐specific microarray. B cells were purified from blood by negative selection; antibody‐producing cells were enumerated by ELISPOT assay. The panel composed of citrulline‐peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline‐peptide panel including the new short epitope peptide of filaggrin, fil311‐315, also identified nearly one‐third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide‐specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline‐containing filaggrin peptides (fil311–315 and fil306–326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline‐peptides of filaggrin and vimentin detect ACPA‐producing cells, and so could also be applied to study the B cells of RA patients.     

Nodular regenerative hyperplasia (NRH) of the liver--a manifestation of 'organ-specific antiphospholipid syndrome'?

Nodular regenerative hyperplasia (NRH) of the liver is a local hyperplastic response of hepatocytes, probably due to vascular abnormalities. Since it was shown in a few case reports that NRH may be associated with antiphospholipid antibodies (APA) we wanted to analyze the relevance of APA in patients with this disease. Sera from 13 patients with histologically defined NRH were tested for APA by an in-house ELISA using as antigens cardiolipin (CL), beta2-glycoprotein I (beta2-gp I), phosphatidylserine (PS), and thromboplastin (TP), a mixture of different phospholipids and phospholipid-binding proteins. As controls, sera from patients with serologically and histologically defined autoimmune liver diseases (primary biliary cirrhosis n = 14; autoimmune hepatitis n = 14) without histological evidence for NRH as well as from 14 healthy blood donors were analyzed. 77% of the NRH patients had APA. In 46% they were directed against CL. In contrast, only 14% of the patients with autoimmune liver diseases and 14% of the healthy controls had anti-CL antibodies (p < 0,05). Antibodies to beta2-gp I and TP did not discriminate between NRH and autoimmune liver diseases. Anti-PS antibodies were not observed. These data indicate that determination of anti-CL antibodies in NRH may help to identify a subgroup of patients in whom an 'organ-specific antiphosholipid syndrome' of the liver may be involved in the pathogenesis.     

Antibody activity against lipopolysaccharides, lipid A and proteins from Enterobacteriaceae in patients with chronic inflammatory liver diseases

These studies are concerned with detection of circulating antibodies against various defined enterobacterial antigens in patients with chronic inflammatory liver diseases such as chronic hepatitis type B (n = 46), chronic active hepatitis (CAH) of autoimmune type (n = 10), alcoholic cirrhosis (n = 24) and primary biliary cirrhosis (PBC) (n = 24) as well as in healthy individuals (n = 39). Anti-LPS and anti-lipid A were determined by hemolytic and hemagglutination assay. Immunoblot technique was used to investigate the antibody activity against plasmid encoded proteins from Yersinia enterocolitica. Persistent titers of anti-LPS up to serum dilution 1:32.768 were found with hemolytic and hemagglutination assay in patients with alcoholic cirrhosis or PBC and in healthy control. In contrast nearly 50% of patients with chronic hepatitis B had no hemolytic antibodies against the two LPS E. coli serotypes at the time of liver biopsy. Anti-lipid A was detectable in 58% of patients with alcoholic cirrhosis but in low titers in less than 10% in the other groups (p less than 0.001). Alcoholic cirrhosis was also associated with a high frequency of IgG and IgA antibodies against plasmid encoded proteins from Yersinia enterocolitica. The data indicate that the O-polysaccharides as strong antigens are physiologically exposed to the immune system while lipid A and enterobacterial proteins are solely immunogenic under abnormal conditions.     

Association of PTPN22 single nucleotide polymorphism with rheumatoid arthritis but not with allergic asthma

PTPN22 gene encodes a lymphoid tyrosine phosphatase (LYP), an important negative regulator of T-cell responses. The 1858C>T (Arg620Trp) single nucleotide polymorphism (rs2476601) was found associated with autoimmune diseases, including rheumatoid arthritis (RA). Allergic diseases are similar to autoimmune diseases, by an exaggerated immune response to an antigen (allergen in this case) normally not invoking such response in healthy individuals. We investigated whether polymorphism 1858C>T in PTPN22 gene is associated with susceptibility to allergic asthma and RA in a Polish population. PTPN22 was genotyped in 173 patients with RA, in 198 patients with allergic asthma, and in 543 controls using PCR-RFLP. The patients with RA differed from healthy controls in frequencies of PTPN22 1858C>T alleles (P=0.0004; odds ratio (OR), 1.8; 95% CI, 1.33-2.55) and genotypes (P=0.0009). Strong associations of 1858T allele with RA limited to joints (0.21 vs 0.12, P=0.0002; OR, 2.1; 95% CI, 1.44-3.00), with erosive disease (0.20 vs 0.12, P=0.0003; OR, 1.92; 95% CI, 1.34-2.71), with a lack of rheumatoid factor (RF; 0.23 vs 0.12, P=0.0008; OR, 2.29; 95% CI, 1.44-3.63), and weak association with the presence of RF (0.17 vs 0.12, P=0.02; OR, 1.6; 95% CI, 1.10-2.40) in comparison with healthy controls were observed. Very strong association of 1858T allele (P<0.0001; OR, 2.72; 95% CI, 1.9-3.9) and T phenotype (P<0001; OR, 3.2; 95% CI, 2.1-4.9) with antibodies to cyclic citrullinated peptide (CCP) was found. When patients with allergic asthma were typed for PTPN22 1858C>T polymorphism, no difference with control was found. Subdivision of patients into those with mild, moderate, or severe asthma did not reveal any associations. In conclusion, we confirmed associations between several clinical manifestations of RA and PTPN22 1858T allele. However, no association with 1858C>T polymorphism was found for susceptibility to allergic asthma or for severity of the disease.     

Centrilobular necrosis in autoimmune hepatitis: a histological feature associated with acute clinical presentation

AIMS: The characteristic histological feature of autoimmune hepatitis (AIH) is interface hepatitis with predominant portal lymphoplasmacytic necroinflammatory infiltration. Centrilobular necrosis (CN), reminiscent of toxic or circulatory liver injury, has been reported in AIH. The aim of this study was to assess the frequency of CN in patients with AIH and its correlation with laboratory and clinical data. METHODS: Liver biopsies were obtained from 114 patients (90 women, 24 men, mean (SD) age 45.4 (19.4) years) with AIH and were evaluated under code by a single pathologist according to the modified Knodell score. RESULTS: CN was found in 20 (17.5%) patients with virtually unaffected portal areas in four cases. Patients with AIH with CN had a higher total hepatic activity index (median (range) 11 (6 to 15) v 5 (2 to 10)) and presented less frequently with cirrhosis (10% v 38%). Patients with CN had a higher frequency of acute onset (87% v 32%), higher bilirubin (median (range) 12.0 (0.43 to 40.0) v 1.9 (0.36 to 46)) and higher ALT levels (median (range) 25.6 (2.7 to 63.9) v 7.2 (0.7 to 62.6)), than did patients with AIH without centrizonal injury. CONCLUSION: CN with sparing of the portal areas represents a rare histological pattern in AIH. CN is associated with an acute clinical presentation and might reflect an early lesion preceding portal involvement. Recognition of this particular histological appearance enables early diagnosis of AIH and a timely initiation of immunosuppressive therapy.     

Fine specificity of anti‐citrullinated peptide antibodies discloses a heterogeneous antibody population in rheumatoid arthritis

Anti‐citrullinated peptide antibodies (ACPA) are highly specific for rheumatoid arthritis (RA). However, the predominant B cell epitopes have not yet been defined. The aim of this study was to examine the reactivity of ACPA against different peptides derived from citrullinated proteins and to investigate whether or not these antibodies constitute a homogeneous population. For this purpose, sera from patients with RA (n = 141), systemic lupus erythematosus (SLE) (n = 60), Sjögren's syndrome (SS) (n = 54) and healthy controls (n = 100) were tested for their reactivity against six citrullinated peptides derived from peptidyl arginine deiminase (PAD), vimentin (vim), alpha‐enolase (enol), fibrin, type II collagen (col‐II) and filaggrin, respectively. A non‐citrullinated control peptide derived from PAD was used as control (ctrlPAD^621–40). Antibody reactivity against each individual peptide was evaluated by enzyme‐linked immunosorbent assay (ELISA). Specificity and cross‐reactivity of ACPA were tested by using two prototype sera with homologous and cross‐inhibition assays. Specificity of ACPA from two prototype sera was confirmed by purification of anti‐peptide antibodies and homologous‐inhibition experiments. We found that sera from patients with RA reacted diversely with the six citrullinated peptides. More specifically, PAD^211–30 displayed 29·08% sensitivity, vim^60–75 29·08%, enol^5–21 37·59%, fibrin^617–31 31·21%, col‐II^358–75 29·97% and filaggrin^306–24 28·37%, while control ctrlPAD^621–40 showed no reactivity. All reactive peptides were found to be highly specific for RA. A notable cross‐reaction (>70%) was found mainly between filaggrin and the majority of anti‐citrullinated peptide antibodies. We concluded that ACPA in RA constitute a heterogeneous population with limited cross‐reactivity and without a predominant epitope.     

Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis

Summary: Rheumatoid arthritis (RA) is now clearly a true autoimmune disease with accumulating evidence of pathogenic disease-specific autoimmunity to citrullinated proteins. Citrullination, also termed deimination, is a modification of arginine side chains catalyzed by peptidylarginine deiminase (PAD) enzymes. This post-translational modification has the potential to alter the structure, antigenicity, and function of proteins. In RA, antibodies to cyclic citrullinated peptides are now well established for clinical diagnosis, though we argue that the identification of specific citrullinated antigens, as whole proteins, is necessary for exploring pathogenic mechanisms. Four citrullinated antigens, fibrinogen, vimentin, collagen type II, and α-enolase, are now well established, with others awaiting further characterization. All four proteins are expressed in the joint, and there is evidence that antibodies to citrullinated fibrinogen and collagen type II mediate inflammation by the formation of immune complexes, both in humans and animal models. Antibodies to citrullinated proteins are associated with HLA ‘shared epitope’ alleles, and autoimmunity to at least one antigenic sequence, the CEP-1 peptide from citrullinated α-enolase (KIHAcitEIFDScitGNPTVE), shows a specific association with HLA-DRB1*0401, *0404, 620W PTPN22, and smoking. Periodontitis, in which Porphyromonas gingivalis is a major pathogenic bacterium, has been linked to RA in epidemiological studies and also shares similar gene/environment associations. This is also the only bacterium identified that expresses endogenous citrullinated proteins and its own bacterial PAD enzyme, though the precise molecular mechanisms of bacterial citrullination have yet to be explored. Thus, both smoking and Porphyromonas gingivalis are attractive etiological agents for further investigation into the gene/environment/autoimmunity triad of RA.     

Diagnostic value of anti-mutated citrullinated vimentin in comparison to anti-cyclic citrullinated peptide and anti-viral citrullinated peptide 2 antibodies in rheumatoid arthritis: An Italian multicentric study and review of the literature

In the last years, the detection of antibodies (Abs) against citrullinated peptides (ACPA) has largely replaced rheumatoid factor (RF) as the most helpful biomarker in the diagnosis of rheumatoid arthritis (RA). Current assays detect ACPA reactivity with epitopes on various different citrullinated proteins. Among these, anti-cyclic citrullinated peptide (CCP) Abs have been widely demonstrated to be an important diagnostic and prognostic tool because of their high specificity. Recently, citrullinated vimentin, a protein highly released in synovial microenvironment, has been identified as potential autoantigen in the pathophysiology of RA and an enzyme-linked immunosorbent assay (ELISA) for the detection of Abs directed against a mutated citrullinated vimentin (anti-MCV) was developed. Several recent studies evaluating the characteristics of anti-MCV in comparison to anti-CCP Abs, have given conflicting results. Anti-MCV have been demonstrated to perform better than anti-CCP as predictor of radiographic damage. Conversely, its additional diagnostic and prognostic role in comparison to anti-CCP in both early and established RA is controversial. Aim of this study was to evaluate the diagnostic performance of anti-MCV in RA and to compare it to anti-CCP and the recently developed assay targeting viral citrullinated peptide 2 (VCP2) in a large cohort of RA patients (n=285), healthy subjects and other disease controls (n=227). Anti-MCV resulted to have a sensitivity of 59% and a specificity of 92%. In comparison, anti-CCP and anti-VCP2 displayed a sensitivity of 77% and 61% and a specificity of 96% and 95%, respectively. Of interest, at the manufacturer recommended cutoff value of 20U/mL, a high percentage of healthy subjects as well as Epstein Barr (EBV) and hepatitis C (HCV) virus infected patients resulted anti-MCV positive. In our large cohort of RA patients, anti-MCV demonstrated lower sensitivity than anti-CCP and VCP2 test, thus not allowing to confirm previously published data. Moreover, the high rate of detection in infectious diseases limits its diagnostic value in undifferentiated arthritis.     

Arthritogenic peptide binding to DRB1*01 alleles correlates with susceptibility to rheumatoid arthritis

Genetic susceptibility to rheumatoid arthritis (RA) is often defined by the presence of a shared epitope (QKRAA, QRRAA, or RRRAA) at positions 70–74 in HLA-DRβ1. However, DRβ1*01:01 and 01:02 contain the same QRRAA epitope, but differ considerably in their susceptibility to RA. The purpose of this study was to determine if this difference could be explained by their ability to bind three arthritogenic peptides that we have previously shown to bind to the archetypal RA-susceptible allele, DRβ1*04:01, but not to the resistant DRβ1*08:01 allele. Binding of type II collagen^258-272, citrullinated and native vimentin^66-78, and citrullinated and native α-enolase^11-25 were measured on cell lines expressing either DRβ1*01:01, *01:02 or *01:03 in association with DRα1*01:01. DRβ1*01:01 and *01:02 both exhibited a 6.5-fold preference for citrullinated vimentin^66-78 compared to native vimentin. However, DRβ1*01:01 also exhibited a 1.7-fold preference for citrullinated α-enolase^11-25 and bound collagen^258-272, while DRβ1*01:02 bound neither of these peptides. Consistent with its known resistance to RA, DRβ1*01:03 preferentially bound native vimentin^66-78 and α−enolase^11-25 over the citrullinated forms of these peptides, and also failed to bind collagen^258−272. Site-directed mutagenesis was performed to determine which amino acid residues were responsible for the differences between these alleles. Mutating position 86 in DRβ1*01:01 from glycine to the valine residue found in DRβ1*01:02 eliminated binding of both citrullinated α-enolase^11-25 and collagen^258-272, thereby recapitulating the peptide-binding profile of DRβ1*01:02. The difference in susceptibility to rheumatoid arthritis between DRβ1*01:01 and *01:02 thus correlates with the effect of position 86 on the binding of these arthritogenic peptides. Consistent with their association with RA resistance, positions I67, D70 and E71 all contributed to the inability of DRβ1*01:03 to bind these arthritogenic peptides.