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1.  Rheumatoid arthritis associated autoantibodies in patients with synovitis of recent onset 
Arthritis Research  2000;2(3):236-243.
An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinically and followed prospectively for 1 year to determine the clinical significance of a number of rheumatoid arthritis (RA) associated autoantibodies. Serum samples collected at the time of the initial evaluation were tested for rheumatoid factor (RF) and antibodies to Sa (anti-Sa), RA-33, (pro)filaggrin [antifilaggrin antibody (AFA)], cyclic citrullinated peptide (anti-CCP), calpastatin, and keratin [antikeratin antibody (AKA)]. RF had a sensitivity of 66% and a specificity of 87% for RA. Anti-Sa, AFA, and anti-CCP all had a specificity of more than 90%, but a sensitivity of less than 50% for this diagnosis. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P < 0.001). Of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one. Finally, anti-SA identified a subset of predominantly male RA patients with severe, erosive disease. Anti-SA, AFA and anti-CCP are all specific for early RA but, overall, have little additional diagnostic value over RF alone. Although these antibodies may preferentially recognize citrullinated antigens, the modest degree of concordance between them in individual patient sera suggests that it is unlikely a single antigen is involved in generating these responses.
Introduction:
A spectrum of autoantibodies is now known to be specifically associated with RA. There continues to be uncertainty as to what stage of the disease each of these autoantibodies develop, and whether they are associated with unique clinical features.
Aims:
To help address these questions, a spectrum of autoantibodies known to be associated with RA in a cohort of patients with early synovitis was evaluated.
Methods:
An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinicially then followed prospectively for 1 year. Patients were classified as having RA on the basis of fulfilling the 1987 criteria. Serum samples collected at the time of the initial evaluation were tested for anti-Sa and anti-RA-33 using immunoblotting, and to (pro)filaggrin (AFA), anti-CCP, and calpastatin (anti-RA-1) using enzyme-linked immunosorbent assay techniques. AKA were detected using immunoflurescence on human epidermal tissue. RF was tested by nephelometry. HLA-DRB1 alleles were determined using sequence specific primers. Initial and 1 year radiographs were evaluated for the presence of erosions.
Results:
Of the 238 patients with synovitis of recent onset in the cohort, 106 (45%) met RA criteria, 102 (96%) of whom met the criteria on their initial visit. Diagnoses in the remaining patients included 22 (9%) with reactive arthritis, 14 (6%) with psoriatic arthritis or another form of spondylarthropathy, 11 (5%) with another well-defined rheumatic diagnosis, and 85 (36%) with undifferentiated arthritis. The RA patients were significantly older than the nonRA patients (46 ± 13 versus 39 ± 13; P < 0.001), had higher mean swollen joint count (13.8 ± 9.7 versus 2.3 ± 2.3; P < 0.001), and higher C-reactive protein (CRP) level (1.9 ± 1.9 versus 1.6 ± 2.4; P < 0.01). Table 1 summarizes the prevalence of the various RA associated antibodies in patients diagnosed as having RF-positive (RF+) RA, RF-negative (RF-) RA, and nonRA. Regarding the characteristics of these tests, RF had the highest sensitivity at 66%, and all the other antibodies individually were less than 50% sensitive. AFA, anti-Sa, anti-CCP were greater than 90% specific for RA, while RF and AKA were 80-90% specific, and anti-RA-33 and anti-RA-1 was not specific for this diagnosis. The data further indicate that adding any one of AFA, AKA, anti-Sa, or anti-CCP to RF increases the specificity for RA from 80 to 90%. In the absence of RF, the presence of one or more of these antibodies carried a sensitivity of only 31% for RF- RA, with anti-Sa being the most specific at 98%. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P < 0.001). Despite this high level of correlation, of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one, suggesting considerable variability in individual reactivity patterns.
RA has been shown in multiple populations to be associated with HLA-DRB1 alleles encoding for the shared epitope (SE). In this study, as illustrated in Table 2, the presence of each of these autoantibodies was significantly associated with having two shared epitope alleles, even when only the RA patients were considered.
Patients with anti-Sa antibodies were predominantly male (61% versus 28%; P<0.01), had significantly higher swollen joint counts (18 ± 12 versus 13 ± 9; P=0.02), and higher CRP levels (2.6 ± 3 mg/dl versus 1.6 ± 1.4 mg/dl; P=0.03) at the initial visit. Despite subsequently begin treated with significantly higher doses of prednisone (4.8 ± 6.0 mg/day versus 1.8 ± 3.3 mg/day; P<0.01), and more disease modifying antirheumatic drug therapy (1.4 ± 0.8 versus 0.9 ± 0.7 disease modifying antirheumatic drugs; P<0.01), the anti-Sa-positive RA patients had a higher frequency of erosions than the rest of the RA patients (60% versus 33%; P=0.03). Neither RF nor SE were associated with the disease severity measures, and analyses evaluating all the other autoantibodies failed to reveal a similar trend.
Discussion:
Despite a well-documented lack of specificity, RF continues to be a central part of the definition of RA, primarily because of its favourable sensitivity profile. In our cohort, RF had a sensitivity of 66%, a specificity of 87%, and an overall accuracy of 78% for the diagnosis of RA. AFA, anti-Sa, anti-CCP were all highly specific for this diagnosis, and when any of them were present in conjunction with RF, the specificity for RA approached 100%. Potentially of more importance to the clinician is the diagnostic value of these antibodies when RF is not detectable. Our data indicate that only 31% of RF- RA patients had any of AKA, AFA, anti-Sa or anti-CCP, and that anti-Sa was the most specific for this diagnosis. This modest level of sensitivity suggests that testing for this spectrum of autoantibodies carries little advantage over RF alone in diagnosing early RA.
AFA, AKA, and antiperinuclear factor (APF) have all been proposed to identify a common antigen present in the skin protein (pro)filaggrin. It has continued to be puzzling why a skin antigen would be targeted relatively specifically in a disorder that is primarily articular. A potential explanation for this may relate to the demonstration that citrulline appears to be an essential constituent of the antigenic determinants recognized by AKA, APF, and AFA. The citrulline rich (pro)filaggrin molecule makes an ideal substrate for detecting this reactivity. Moreover, the SA antigen, which, unlike (pro)filaggrin, is detectable in rheumatoid synovium, has recently been shown to also be citrullinated. It is thus possible that AKA, AFA, APE, and anti-Sa all recognize one or more citrullinated antigens. Despite this possibility, the modest degree of concordance between them in individual patient sera suggests that it is unlikely that a single antigen is involved in generating these responses.
This study provides evidence suggesting that anti-Sa antibodies appear to be a marker for a subset of early RA patients whose disease may be more severe and erosive. Moreover, it was determined that anti-Sa, AFA, and anti-CCP were all highly associated with SE, particularly two copies. We examined a spectrum of potential RA severity indicators including the number of swollen joints, CRP level, and presence of early radiographic erosions. Our data indicate that anti-Sa was more highly associated with these measures of RA severity than any other parameter, including the most accepted prognostic indicators, RF and SE.
In conclusion, it is demonstrated that antibodies directed against putatively citrullinated antigens including SA, filaggrin, keratin, and CCP are the most specific for RA, and are detectable early in the disease course. It will be of interest to find out whether the cumulative prevalence of specific autoantibody subsets tends to increase over time, as this would suggest that the mechanisms underlying the development of these reactivities continue to evolve over the course of the arthropathy.
PMCID: PMC17811  PMID: 11056669
autoantibodies; early synovitis; human leukocyte antigen; rheumatoid arthritis; spondylarthropathy
2.  Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid Synovium 
PLoS Medicine  2009;6(1):e1.
Background
Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.
Methods and Findings
Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.
Conclusions
Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies.
Costantino Pitzalis and colleagues show that lymphoid structures in synovial tissue of patients with rheumatoid arthritis support production of anti-citrullinated peptide antibodies, which continues following transplantation into SCID mice.
Editors' Summary
Background.
More than 1 million people in the United States have rheumatoid arthritis, an “autoimmune” condition that affects the joints. Normally, the immune system provides protection against infection by responding to foreign antigens (molecules that are unique to invading organisms) while ignoring self-antigens present in the body's own tissues. In autoimmune diseases, this ability to discriminate between self and non-self fails for unknown reasons and the immune system begins to attack human tissues. In rheumatoid arthritis, the lining of the joints (the synovium) is attacked, it becomes inflamed and thickened, and chemicals are released that damage all the tissues in the joint. Eventually, the joint may become so scarred that movement is no longer possible. Rheumatoid arthritis usually starts in the small joints in the hands and feet, but larger joints and other tissues (including the heart and blood vessels) can be affected. Its symptoms, which tend to fluctuate, include early morning joint pain, swelling, and stiffness, and feeling generally unwell. Although the disease is not always easy to diagnose, the immune systems of many people with rheumatoid arthritis make “anti-citrullinated protein/peptide antibodies” (ACPA). These “autoantibodies” (which some experts believe can contribute to the joint damage in rheumatoid arthritis) recognize self-proteins that contain the unusual amino acid citrulline, and their detection on blood tests can help make the diagnosis. Although there is no cure for rheumatoid arthritis, the recently developed biologic drugs, often used together with the more traditional disease-modifying therapies, are able to halt its progression by specifically blocking the chemicals that cause joint damage. Painkillers and nonsteroidal anti-inflammatory drugs can reduce its symptoms, and badly damaged joints can sometimes be surgically replaced.
Why Was This Study Done?
Before scientists can develop a cure for rheumatoid arthritis, they need to know how and why autoantibodies are made that attack the joints in this common and disabling disease. B cells, the immune system cells that make antibodies, mature in structures known as “germinal centers” in the spleen and lymph nodes. In the germinal centers, immature B cells are exposed to antigens and undergo two genetic processes called “somatic hypermutation” and “class-switch recombination” that ensure that each B cell makes an antibody that sticks as tightly as possible to just one antigen. The B cells then multiply and enter the bloodstream where they help to deal with infections. Interestingly, the inflamed synovium of many patients with rheumatoid arthritis contains structures that resemble germinal centers. Could these ectopic (misplaced) lymphoid structures, which are characterized by networks of immune system cells called follicular dendritic cells (FDCs), promote autoimmunity and long-term inflammation by driving the production of autoantibodies within the joint itself? In this study, the researchers investigate this possibility.
What Did the Researchers Do and Find?
The researchers collected synovial tissue from 55 patients with rheumatoid arthritis and used two approaches, called immunohistochemistry and real-time PCR, to investigate whether FDC-containing structures in synovium expressed an enzyme called activation-induced cytidine deaminase (AID), which is needed for both somatic hypermutation and class-switch recombination. All the FDC-containing structures that the researchers found in their samples expressed AID. Furthermore, these AID-containing structures were surrounded by mature B cells making ACPAs. To test whether these B cells were derived from AID-expressing cells resident in the synovium rather than ACPA-expressing immune system cells coming into the synovium from elsewhere in the body, the researchers transplanted synovium from patients with rheumatoid arthritis under the skin of a special sort of mouse that largely lacks its own immune system. Four weeks later, the researchers found that the transplanted human lymphoid tissue was still making AID, that the level of AID expression correlated with the amount of human ACPA in the blood of the mice, and that the B cells in the transplant were proliferating.
What Do These Findings Mean?
These findings show that the ectopic lymphoid structures present in the synovium of some patients with rheumatoid arthritis are functional and are able to make ACPA. Because ACPA may be responsible for joint damage, the survival of these structures could, therefore, be involved in the development and progression of rheumatoid arthritis. More experiments are needed to confirm this idea, but these findings may explain why drugs that effectively clear B cells from the bloodstream do not always produce a marked clinical improvement in rheumatoid arthritis. Finally, they suggest that AID might provide a new target for the development of drugs to treat rheumatoid arthritis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0060001.
This study is further discussed in a PLoS Medicine Perspective by Rene Toes and Tom Huizinga
The MedlinePlus Encyclopedia has a page on rheumatoid arthritis (in English and Spanish). MedlinePlus provides links to other information on rheumatoid arthritis (in English and Spanish)
The UK National Health Service Choices information service has detailed information on rheumatoid arthritis
The US National Institute of Arthritis and Musculoskeletal and Skin Diseases provides Fast Facts, an easy to read publication for the public, and a more detailed Handbook on rheumatoid arthritis
The US Centers for Disease Control and Prevention has an overview on rheumatoid arthritis that includes statistics about this disease and its impact on daily life
doi:10.1371/journal.pmed.0060001
PMCID: PMC2621263  PMID: 19143467
3.  Autoantibodies and autoantigens in autoimmune hepatitis: important tools in clinical practice and to study pathogenesis of the disease 
Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver characterized by hypergammaglobulinemia, characteristic autoantibodies, association with HLA DR3 or DR4 and a favorable response to immunosuppressive treatment. The etiology is unknown. The detection of non-organ and liver-related autoantibodies remains the hallmark for the diagnosis of the disease in the absence of viral, metabolic, genetic, and toxic etiology of chronic hepatitis or hepatic injury. The current classification of AIH and the several autoantibodies/target-autoantigens found in this disease are reported. Current aspects on the significance of these markers in the differential diagnosis and the study of pathogenesis of AIH are also stated. AIH is subdivided into two major types; AIH type 1 (AIH-1) and type 2 (AIH-2). AIH-1 is characterized by the detection of smooth muscle autoantibodies (SMA) and/or antinuclear antibodies (ANA). Determination of antineutrophil cytoplasmic autoantibodies (ANCA), antibodies against the asialoglycoprotein receptor (anti-ASGP-R) and antibodies against to soluble liver antigens or liver-pancreas (anti-SLA/LP) may be useful for the identification of patients who are seronegative for ANA/SMA. AIH-2 is characterized by the presence of specific autoantibodies against liver and kidney microsomal antigens (anti-LKM type 1 or infrequently anti-LKM type 3) and/or autoantibodies against liver cytosol 1 antigen (anti-LC1). Anti-LKM-1 and anti-LKM-3 autoantibodies are also detected in some patients with chronic hepatitis C (HCV) and chronic hepatitis D (HDV). Cytochrome P450 2D6 (CYP2D6) has been documented as the major target-autoantigen of anti-LKM-1 autoantibodies in both AIH-2 and HCV infection. Recent convincing data demonstrated the expression of CYP2D6 on the surface of hepatocytes suggesting a pathogenetic role of anti-LKM-1 autoantibodies for the liver damage. Family 1 of UDP-glycuronosyltransferases has been identified as the target-autoantigen of anti-LKM-3. For these reasons the distinction between AIH and chronic viral hepatitis (especially of HCV) is of particular importance. Recently, the molecular target of anti-SLA/LP and anti-LC1 autoantibodies were identified as a 50 kDa UGA-suppressor tRNA-associated protein and a liver specific enzyme, the formiminotransferase cyclodeaminase, respectively. Anti-ASGP-R and anti-LC1 autoantibodies appear to correlate closely with disease severity and response to treatment suggesting a pathogenetic role of these autoantibodies for the hepatocellular injury. In general however, autoantibodies should not be used to monitor treatment, predict AIH activity or outcome. Finally, the current aspects on a specific form of AIH that may develop in some patients with a rare genetic syndrome, the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) are also given. Autoantibodies against liver microsomes (anti-LM) are the specific autoantibodies detected in AIH as a disease component of APECED but also in cases of dihydralazine-induced hepatitis. Cytochrome P450 1A2 has been identified as the target-autoantigen of anti-LM autoantibodies in both APECED-related AIH and dihydralazine-induced hepatitis. The latter may indicate that similar autoimmune pathogenetic mechanisms can lead to liver injury in susceptible individuals irrespective of the primary defect. Characterization of the autoantigen-autoantibody repertoire continues to be an attractive and important tool to get access to the correct diagnosis and to gain insight into the as yet unresolved mystery of how hepatic tolerance is given up and AIH ensues.
doi:10.1186/1740-2557-1-2
PMCID: PMC544946  PMID: 15679907
Antibodies against Liver Cytosol 1 Antigen (anti-LC1); Antibodies against Soluble Liver Antigens or Liver Pancreas (anti-SLA/LP); Antinuclear Antibodies (ANA); Antineutrophil Cytoplasmic Autoantibodies (ANCA); Autoimmune Hepatitis; Cytochrome P450 2D6; Cytochrome P450 2A6; Cytochrome P450 1A2; Hepatitis C; Hepatitis D; Liver-Kidney Microsomal Autoantibodies (anti-LKM); Liver Microsomal Autoantibodies (anti-LM); Smooth Muscle Autoantibodies (SMA)
4.  Anti-perinuclear factor compared with the so called "antikeratin" antibodies and antibodies to human epidermis filaggrin, in the diagnosis of arthritides 
OBJECTIVE—Antiperinuclear factor (APF), "antikeratin antibodies" ("AKA"), and antibodies to human epidermis filaggrin (AFA), are highly specific serological markers of rheumatoid arthritis (RA), which recognise epitopes on various isoforms of (pro)filaggrin. It was proposed that these antibodies are globally named antifilaggrin autoantibodies. Here the diagnostic value of the detection of each one is compared and the overlap between the three tests evaluated.
METHODS—492 serum samples were tested, including 279 RA serum samples, taken from patients in France and Belgium. APF and "AKA" titres were estimated by indirect immunofluorescence, and AFA titres by immunoblotting on filaggrin enriched human epidermis extracts.
RESULTS—By a convenient choice of the positivity thresholds, the diagnostic sensitivity and specificity of the tests were shown to be similar (0.52 and 0.97, respectively). Although the antibody titres were strongly correlated, the associations APF-AFA or AFA-"AKA" permitted more than 52% or 55% of RA to be diagnosed, with a specificity of 0.99.
CONCLUSION—APF, "AKA", and AFA detection have a similar diagnostic value. However, because the three tests do not totally overlap, associating APF with "AKA" or AFA with "AKA" can improve diagnostic sensitivity. None of the three antigens used bear all the epitopes recognised by anti-filaggrin autoantibodies.

 Keywords: rheumatoid arthritis; antifilaggrin autoantibodies; antiperinuclear factor; antikeratin antibodies
PMCID: PMC1752764  PMID: 10343539
5.  Expression of the Inherently Autoreactive Idiotope 9G4 on Autoantibodies to Citrullinated Peptides and on Rheumatoid Factors in Patients with Early and Established Rheumatoid Arthritis 
PLoS ONE  2014;9(9):e107513.
Background
The pre-symptomatic stage of Rheumatoid arthritis (RA) is associated with pro-inflammatory cytokines and autoantibodies. High levels and epitope spread by Rheumatoid factors (RhF) and autoantibodies to citrullinated proteins signify progression towards disease expression. In established RA, the persistence of high autoantibody levels reflects production by both long-lived plasma cells and short-lived plasmablasts. Neither the relative contributions to pathogenesis by autoantibodies from either source, nor the factors responsible for deciding the fate of autoantigen specific ‘parent’ B-cells, is understood. Phenotypic markers identifying subsets of autoreactive B-cells are therefore of interest in understanding the origin and perpetuation of the autoimmune response in RA. One such phenotypic marker is the rat monoclonal antibody, 9G4, which recognises an idiotope on immunoglobuins derived from the inherently autoreactive VH-gene, VH4-34. We therefore investigated whether the 9G4 idiotope was expressed on autoantibodies in patients with RA.
Methodology/Principal Findings
Sera from 19 patients with established RA and those with <1year history of untreated polyarthritis either resolving into RA (n = 42) or non-RA diagnosis (n = 31) were included. Autoantibodies to cyclic citrullinated peptides (CCP), RhF and co-expression of the 9G4 idiotope were measured by ELISA. 9G4 recognised a population of anti-CCP antibodies in the majority of sera from patients with established disease and also in samples from patients with early disaese. 9G4+RhF levels were generally lower and not associated with positivity for, or levels of 9G4+CCP.
Conclusions/Significance
The persistence of 9G4+ immunoglobulins, of any isotype, in serum is rare. We describe here the novel finding of 9G4 expression on anti-CCP antibodies in patients from the earliest symptoms of RA through to established disease. Our results suggest that 9G4 expression on anti-CCP autoantibodies was not due to polyclonal expansion of VH4-34-encoded immunoglobulins. These studies may therefore provide a new focus for investigation into the evolution of the autoimmune response in RA patients.
doi:10.1371/journal.pone.0107513
PMCID: PMC4164660  PMID: 25222933
6.  Epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination 
Arthritis Research & Therapy  2008;10(5):R119.
Introduction
Anti-citrullinated protein antibodies have a diagnostic role in rheumatoid arthritis (RA); however, little is known about their origins and contribution to pathogenesis. Citrullination is the post-translational conversion of arginine to citrulline by peptidyl arginine deiminase, and increased citrullination of proteins is observed in the joint tissue in RA and in brain tissue in multiple sclerosis (MS).
Methods
We applied synovial and myelin protein arrays to examine epitope spreading of B cell responses to citrullinated epitopes in both the collagen-induced arthritis (CIA) model for RA and the experimental autoimmune encephalomyelitis (EAE) model for MS. Synovial and myelin protein arrays contain a spectrum of proteins and peptides, including native and citrullinated forms, representing candidate autoantigens in RA and MS, respectively. We applied these arrays to characterise the specificity of autoantibodies in serial serum samples derived from mice with acute and chronic stages of CIA and EAE.
Results
In samples from pre-disease CIA and acute-disease EAE, we observed autoantibody targeting of the immunising antigen and responses to a limited set of citrullinated epitopes. Over the course of diseases, the autoantibody responses expanded to target multiple citrullinated epitopes in both CIA and EAE. Using immunoblotting and mass spectrometry analysis, we identified citrullination of multiple polypeptides in CIA joint and EAE brain tissue that have not previously been described as citrullinated.
Conclusions
Our results suggest that anti-citrulline antibody responses develop in the early stages of CIA and EAE, and that autoimmune inflammation results in citrullination of joint proteins in CIA and brain proteins in EAE, thereby creating neoantigens that become additional targets in epitope spreading of autoimmune responses.
doi:10.1186/ar2523
PMCID: PMC2592807  PMID: 18826638
7.  Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods 
Arthritis Research  2000;2(5):407-414.
The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities.
Introduction:
The classification of antinuclear antibodies (ANAs) is important for diagnosis and prognosis and for understanding the molecular pathology of autoimmune disease. Many of the proteins that associate with RNA in the ribonucleoprotein (RNP) complexes of the spliceosome have been found to react with some types of ANA [1], including proteins of the heterogeneous nuclear RNP (hnRNP) complex that associate with newly transcribed pre-mRNA. Autoantibodies to the A2, B1, and B2 proteins of hnRNP found in some patients may be markers of several overlap syndromes [2]. However, ANAs with specificity for these proteins as well as for the D protein also appear to occur in many distinct connective-tissue diseases, although epitope specificities may differ [3]. ANAs with specificity for the C component of hnRNP (consisting of the C1 and C2 proteins) have to our knowledge so far been described in only one case [4]. We here describe the approach taken to unambiguously identify the C1/C2 proteins as ANA targets in the sera of some patients.
Aims:
To determine the fine specificity of sera containing an unusual speckled ANA-staining pattern using a combination of 2D gel electrophoresis and MS.
Methods:
Patient sera were screened for ANAs by indirect immunofluorescence microscopy on HEp-2 cells (cultured carcinoma cells). Sera with an unusual, very regular, speckled ANA pattern were tested for reactivity with components of nuclear extracts of HeLa cells that were separated by one-dimensional (1D) or 2D gel electrophoresis or by reversed-phase high-performance liquid chromatography (HPLC). IgG reactivity was assessed by immunoblotting. Reactive protein spots from 2D separations were excised from the gels and subjected to in-gel digestion with trypsin for subsequent peptide mapping, partial peptide sequencing, and protein identification by MS and tandem MS on a hybrid electrospray ionization/quadrupole/time-of-flight (ESI-Q-TOF) mass spectrometer [5,6,7].
Results:
We observed a strong nuclear staining pattern (titer >1280) with the characteristic even-sized coarse speckles and no staining of nucleoli in sera from three patients. On immunoblots of nuclear extracts from HeLa cells, these sera stained two distinct bands, at Mr 42 000 and 41 000. There activity strongly resembled that of the patient originally described by Stanek et al [4]. The antigens were enriched by fractionating the extract using reversed-phase HPLC on a C4 column, and the two reactive spots on 2D separations were excised for identification. The two components appeared to be of approximately the same isoelectric points, although their molecular masses differed by approximately 2000. Peptide-mass mapping was performed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS on the tryptic peptide mixture generated by digestion of the two excised proteins. The database search suggested that the two proteins were C1/C2 hnRNPs (Swissprot accession number P07910). The identity of the proteins was further confirmed by tandem MS using an ESI-Q-TOF instrument. One peptide carrying two positive charges (m/z 580.32 Da), corresponding to a peptide mass of 1158.7 Da, was selected as a precursor ion and partially sequenced by collisional fragmentation. The fragmented peptide was found to represent the tryptic fragment VDSLLENLEK, ie amino acids 207-216 (C2 protein numbering). Four other peptides were partially sequenced and all of them matched the human C1/C2 hnRNP sequence. The theoretical masses of C1 and C2 are 32.0 and 33.3 kDa, respectively. The difference between the two sequences is a 13-amino-acid insert in C2 between positions 107 and 108 of C1. The presence of a specific tryptic fragment in the MALDI-TOF peptide-mass map from the higher-molecular-mass spot containing a 13-amino-acid insert that was not present in the lower-molecular-mass spot, further demonstrated that the two components represented the two isoforms of the C class of hnRNPs.
The patient whose case prompted us to investigate the specificities of these antibodies was a 72-year-old man who had arthralgias and oligoarthritis but did not fulfill the criteria for rheumatoid arthritis and did not have dermatological complaints. The reactivity of various patient groups to the C1/C2 hnRNP autoantigens was subsequently tested by immunoblotting of HeLa-cell nuclear extracts. Of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis, none had IgG antibodies reacting with the two bands. Of sera from 139 consecutive patients who had moderately to strongly positive speckled ANA patterns shown by indirect immunofluorescence on HEp-2 cells, only two reacted with the C1/C2 hnRNP bands in immunoblotting. One of these was from a young woman (22 years old) whose complaints of muscle tenderness were not explained by objective findings or abnormal laboratory test results. The third patient that we identified through ANA screening followed by immunoblotting was a 54-year-old male who was being treated with methotrexate for long-standing polymyositis in addition to psoriasis and possible osteoporosis.
Discussion:
The results confirm the existence of anti-C1/C2 antibodies in some patients with speckled ANAs. The antigens were identified through the use of biochemical methods using high-resolution separation techniques combined with mass-spectrometry peptide mapping and database searches. As a general approach, this is a powerful way to identify new antigens using small amounts of material without the need for conventional protein sequencing. The approach does require, however, that the proteins can be found in databases, that they are not extensively post-translationally modified, that they can be digested enzymatically, and that they can be isolated in appropriately pure form by the separation technique used.
It is not known at present if the C1/C2 antibodies may have pathogenic relevance and/or relate to specific diagnoses or subsets within the group of connective-tissue diseases. It does appear that the reactivity is quite rare among ANA-positive patients, and therefore many patients will have to be examined to determine these issues. The fact that the antibodies to the C1/C2 hnRNPs are revealed by indirect immunofluorescence would indicate that the epitopes are accessible in intact, fixed HEp-2 cells and thus probably reside outside the nucleic-acid-binding domains that would be expected to be covered by RNA.
PMCID: PMC17817  PMID: 11056675
antinuclear antibodies; autoantibodies; heterogeneous nuclear ribonucleoproteins C1/C2; mass spectrometry
8.  P53 autoantibodies in 1006 patients followed up for breast cancer 
Breast Cancer Research : BCR  2000;2(6):438-443.
Serial plasma samples from 1006 patients with breast cancer revealed: (i) no correlation of p53 autoantibody status with disease status at the time of sample collection, or with menopausal status at time of primary diagnosis of breast cancer; (ii) 155 out of 1006 (15%) of patients were positive for p53 autoantibodies, and these patients tended to have a persistent autoantibody status throughout follow up, irrespective of disease behaviour; and (iii) where a negative autoantibody status was found at primary diagnosis of breast cancer, this negative status persisted throughout follow up, irrespective of later disease behaviour. We conclude that screening for p53 autoantibody status is not informative on residual tumour activity nor on therapeutic responsiveness.
Introduction:
Dysfunction of the tumour-suppressor protein, p53, may be due to either mutational or epigenetic factors, each of which may lead to accumulation of cytoplasmic p53. Abnormal accumulation of p53 in breast cancer tissue is predictive of poor prognosis [1,2]. Humoral studies [3,4] have shown that cancer patients may develop immunity to abnormally expressed p53, as revealed by p53 autoantibodies in the blood. Again, prognostic correlates have been noted, with presence of circulating p53 autoantibodies at diagnosis of breast cancer being associated with reduced overall survival [5,6] and with poor prognostic factors such as high histological grade and the absence of hormone receptors [5,7,8].
Little is known of the potential value of p53 autoantibody in follow up of cancer. In lung cancer there is evidence that autoantibodies to p53 may provide a useful tool to monitor response to therapy [9,10], whereas serial measurements of autoantibodies to p53 in 40 patients with advanced ovarian cancer were not found to be clinically useful [11]. In breast cancer some 30% of node-negative patients will relapse within 5 years, but there is no current means to predict those who are at risk.
We performed the present study to ask if the presence of autoantibodies to p53 has any association with breast cancer progression.
Materials and method:
A library of plasma samples were collected from all patients attending one general oncology clinic for postoperative follow up of breast cancer. The clinical status of each patient at the time of sampling was summarized. An average of eight plasma samples were cryopreserved for each patient over a period of 15 years.
The enzyme-linked immusorbent assay (ELISA) for p53 autoantibodies was developed in-house, based on the ELISA procedure of Lubin et al [3]. Our in-house method is detailed in the full text of this article. In one assay series we compared a commercial ELISA kit for p53 autoantibodies with our in-house ELISA. A total of 20 patients' samples were tested, representing a range of positive and negative readings. Two samples scored as strongly positive with the in-house assay, but only one of these two scored positive with the commercial assay. Having established sensitivity, specificity and reproducibility of the in-house assay, we judged that this was superior to the commercial assay both in terms of sensitivity and of cost (£1 per test compared with £23 per test). The in-house assay was thus used throughout the present study.
Results:
Serial plasma samples from 1006 patients with breast cancer revealed the following: (i) no correlation of p53 autoantibody status with disease status at the time of sample collection (Table 1), or with menopausal status at time of primary diagnosis of breast cancer (Table 2); (ii) 155 out of 1006 (15%) of patients were positive for p53 autoantibodies, and these patients tended to have a persistent autoantibody status throughout follow up, irrespective of disease behaviour; and (iii) where a negative autoantibody status was found at primary diagnosis of breast cancer, this negative status persisted throughout follow up, irrespective of later disease behaviour (Table 3).
Discussion:
As a working hypothesis, we proposed that levels of autoantibodies to p53 would reflect tumour behaviour. However, we found that the presence or absence of p53 autoantibodies was not predictive of presence or absence of recurrent disease. There was an equivalent incidence of active disease at the time of sampling in both the autoantibody-negative and autoantibody-positive groups, these being 25.2 and 28.7%, respectively. Thus, humoral immune activity against p53 appeared to be relatively restricted to a subgroup of patients in whom, once an autoantibody response had been generated, antibody was likely to persist regardless of tumour behaviour. Conversely, where no detectable p53 autoantibody was present at the time of primary diagnosis, these patients remained similarly negative for antibody, irrespective of subsequent disease activity (Table 3).
In contrast to shed markers that correlate with tumour mass, such as CA15.3 for cancer of the breast, any tumour-related immune response will be subject to complex regulation. Autoantibody responses to p53 will require appropriate primary immunization; initial low-dose antigen exposure may induce immune tolerance and lack of response. Higher antigen doses may activate either antibody-mediated immunity, or cellular immunity.
In breast cancer patients, our results suggest that, once an active humoral response against p53 is established, then this remains active. This persistent humoral reaction may be driven by persistent antigenic stimulation by p53 protein derived from overexpression of p53 at distant metastatic sites; alternatively, irradiated normal tissue may be a source of continued antigenic stimulation, because a long-term side effect of radiation therapy is an increased expression of p53 in normal breast tissue that persists for several years [12]. Since the great majority of our total patient cohort had received radiotherapy, humoral immunity to p53 associated with primary disease might persist, even in those patients who enter remission, due to tumour-independent antigenic stimulation.
Loss of p53 function is known to correlate with loss of efficacy of cancer therapy in vivo [13,14]. This raised the possibility that autoantibodies to p53 that develop during follow up might indicate those patients whose tumor has become resistant to therapy. However, the present results show that, if no immunity has been generated at the time of primary diagnosis, then later immunity is unlikely to occur. This corresponds to the finding that expression of p53 antigen in biopies of locally advanced breast cancer did not correlate with drug resistance [15,16]. Overall, the present observations show that screening for p53 autoantibody status is not informative on residual tumour activity, or on therapeutic responsiveness. We conclude that the potential value of p53 autoantibody screening in patients with breast cancer is limited to the prognostic information obtained at diagnosis.
PMCID: PMC13921  PMID: 11056691
breast; cancer; monitoring; p53 autoantibodies
9.  The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis. 
Journal of Clinical Investigation  1993;92(3):1387-1393.
In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneum of rat esophagus epithelium has been widely reported. These so-called "antikeratin antibodies," detected by indirect immunofluorescence, were found to be autoantibodies since they also labeled human epidermis. Despite their name, the actual target of these autoantibodies was not known. In this study, a 40-kD protein (designated as 40K), extracted from human epidermis and specifically immunodetected by 75% of RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin, a cytokeratin filament-aggregating protein, by peptide mapping studies and by the following evidences: (a) mAbs specific for filaggrin reacted with the 40K protein; (b) the autoantibodies, affinity-purified from RA sera on the 40K protein, immunodetected purified filaggrin; (c) the reactivity of RA sera to the 40K protein was abolished after immunoadsorption with purified filaggrin; (d) the 40K protein and filaggrin had similar amino acid compositions. Furthermore, autoantibodies against the 40K protein and the so-called "antikeratin antibodies" were shown, by immunoadsorption experiments, to be largely the same. The identification of filaggrin as a RA-specific autoantigen could contribute to the understanding of the pathogenesis of this disease and, ultimately, to the development of methods for preventing the autoimmune response.
Images
PMCID: PMC288281  PMID: 7690781
10.  Autoantibody and idiotype profile of lung involvement in autoimmune rheumatic disease. 
Annals of the Rheumatic Diseases  1990;49(3):160-162.
Several reports have linked the presence of certain serum autoantibodies with particular clinical manifestations of autoimmune disease. For example, the Jo-1 antibody is now established as a marker for fibrosing alveolitis in polymyositis. To investigate the possible association of further autoantibodies or idiotypes with fibrosing alveolitis in autoimmune rheumatic disease a panel of autoantibodies was measured in serum samples from 28 patients with systemic lupus erythematosus (SLE) (10 with and 18 without lung involvement), 21 patients with scleroderma (12 with fibrosing alveolitis and nine without), and 41 patients with 'lone' fibrosing alveolitis. Antibodies measured were IgM and IgG anti-dsDNA and anti-ssDNA antibody; IgG and IgM anticardiolipin antibody; anti-poly (ADP-ribose) antibody; antibodies to two common idiotypes of anti-DNA antibodies, designated 134 and 16/6; and IgM, IgG, and IgA isotypes of rheumatoid factor. None of these antibodies was specifically associated with lung involvement in SLE or scleroderma, but a trend was found towards an increase in all autoantibodies in association with lung disease in SLE, while the reverse trend was seen in scleroderma.
PMCID: PMC1004012  PMID: 2322027
11.  Autoimmunity against Fibrinogen Mediates Inflammatory Arthritis in Mice 
Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the presence of anticitrullinated protein Abs, although the exact targets and role of anticitrullinated protein autoimmunity in the pathogenesis of RA remain to be defined. Fibrinogen, which can be citrullinated, has recently emerged as a candidate autoantigen. To determine whether autoimmunity against fibrinogen can mediate inflammatory arthritis, we immunized a variety of common mouse strains with fibrinogen and found that DBA/1 and SJL mice developed an inflammatory and erosive arthritis. Mice with fibrinogen-induced arthritis (FIA) possess fibrinogen-reactive T cells that produce the proinflammatory cytokines IL-6, IL-17, TNF-α, and IFN-γ. FIA can be adoptively transferred with either plasma or fibrinogen-specific T cells from diseased mice. Mice with FIA possess rheumatoid factor, circulating immune complexes, and anticyclic citrullinated peptide Abs, all of which are characteristic of human RA. These observations demonstrate that fibrinogen is arthritogenic in mice and that the pathogenesis of FIA is mediated by both autoantibodies and fibrinogen-reactive T cells.
doi:10.4049/jimmunol.0901639
PMCID: PMC3412066  PMID: 19949094
12.  Risk factors for ANA positivity in healthy persons 
Introduction
The finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus.
Methods
Biological samples from healthy individuals and patients with systemic lupus erythematosus (SLE) were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an array with more than 100 specificities. Whole blood RNA samples from a subset of individuals were used to analyze gene expression on the Illumina platform. Data were analyzed for associations of high ANA levels with demographic features, the presence of other autoantibodies and with gene expression profiles.
Results
Overall, ANA levels are significantly higher in females than in males and this association holds in patients with the autoimmune diseases lupus and rheumatoid arthritis (RA) as well as in healthy controls (HC). Age was not significantly associated with ANA levels and the elevated ANA values could not be explained by higher IgG levels. Another autoantibody, anti- cyclic citrullinated peptide (CCP), did not show gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in high ANA HCs. Some of these autoantibodies were directed to antigens in skin and others were related to autoimmune conditions of kidney, thyroid or joints. Gene expression analyses showed a greater prevalence of significantly upregulated genes in HCs with negative ANA values than in those with significant ANA positivity. Genes upregulated in high ANA HCs included a celiac disease autoantigen and some components of the Type I interferon (IFN) gene signature.
Conclusions
Risks for ANA positivity include female gender and organ-specific autoimmunity. Upregulation of skin-specific autoantibodies may indicate that early events in the break of tolerance take place in cutaneous structures. Some of these changes may be mediated by Type I IFN. Blood profiling for expressed autoantibodies and genes has the potential to identify individuals at risk for development of autoimmune diseases including lupus.
doi:10.1186/ar3271
PMCID: PMC3132017  PMID: 21366908
13.  Unique Correlation Between Mutated Citrullinated Vimentine IgG Autoantibodies and Markers of Systemic Inflammation in Rheumatoid Arthritis Patients 
Rheumatoid arthritis (RA) is the most common inflammatory systemic autoimmune disease, primarily affecting the peripheral joints. Anti-mutated citrullinated vimentin autoantibodies (anti-MCV) of IgG isotype were shown to be a useful diagnostic marker of RA especially in RA patients who were anti-cyclic citrullinated protein autoantibodies (anti-CCP) negative. Nevertheless, published data correlates rheumatoid factor (RF), anti-CCP or anti-MCV antibodies with either erythrocyte sedimentation rate (ESR) or serum C-reactive protein (CRP) as markers of disease activity, not investigated the possible correlations of RA autoantibodies towards ESR and CRP in comparison. Herein, we aim to evaluate the usefulness of anti-MCV as a dependable marker in established RA compared with anti-CCP and RF antibodies and to examine correlations between RF, anti-CCP and anti-MCV antibodies towards ESR and serum CRP. Serum RF-IgA, RF-IgM, anti-CCP and anti-MCV levels were measured in 30 patients with RA and 40 patients with other autoimmune diseases (non-RA) compared with 20 normal subjects. Specificity, sensitivity and AUC for RF antibodies, anti-CCP and anti-MCV were calculated towards RA diagnosis. Our results showed that ESR and CRP had significantly higher values in both RA and non-RA patients compared with our healthy controls with observed significant increment in RA patients compared with non-RA patients. An important finding from our study is that 33.3 % of RA patients were anti-CCP negative but being positive towards anti-MCV. Also, in-between 36.7 up to 40 % of RA patients were RF-IgA and RF-IgM negative while being anti-MCV positive. Anti-MCV antibodies showed the highest specificity and sensitivity (97.5 and 86.6 %, respectively) towards RA diagnosis with the highest AUC value (0.920) compared with anti-CCP and RF antibodies. Correlation analyses revealed that there was no significant correlation between ESR along with CRP towards RF-IgA, RF-IgM and anti-CCP while profound highly significant correlation exhibited between ESR and CRP towards anti-MCV data (r = 0.879 and 0.994, respectively). Thus, our data suggest that the assessment of serum anti-MCV autoantibodies along with ESR and CRP considered as a simple laboratory regime for monitoring RA patients to assess and follow-up disease activity. The addition of anti-MCV autoantibodies to serologic markers in the ACR/EULAR classification criteria for RA will add points for patients with negative anti-CCP and RF antibodies.
doi:10.1007/s12291-012-0272-1
PMCID: PMC3689334  PMID: 24426223
Rheumatoid arthritis; Anti-mutated citrullinated vimentine antibodies; Specificity; Sensitivity; C-reactive protein; Erythrocyte sedimentation rate
14.  Invariant Natural Killer T Cells Inhibit Autoreactive B Cells in a Contact- and CD1d-dependent Manner 
Autoantibody production is a hallmark of autoimmune diseases such as lupus and rheumatoid arthritis. Accumulating evidence suggests a role of invariant natural killer T cells (iNKT) in their pathogenesis. Mechanisms underlying iNKT role in these diseases, however, remain unclear. Here, we show that iNKTs suppress IgG anti-DNA Ab and rheumatoid factor production and reduce interleukin-10–secreting B cells in a contact-dependent manner, but increase total IgG production and enhance activation markers on B cells via soluble factors. In vivo reconstitution with iNKTs also reduces autoantibody production in iNKT-deficient mice and in SCID mice implanted with B cells. Using an anti-DNA transgenic model, we found that autoreactive B cells spontaneously produce IL-10 and are activated in vivo. In the presence of activated iNKTs, these autoreactive B cells are selectively reduced, whereas non-autoreactive B cells are markedly activated. Since iNKTs recognize CD1d, we reasoned that CD1d might play a role in the differential regulation of autoreactive vs. non-autoreactive B cells by iNKTs. Indeed, autoreactive B cells express more CD1d than non-autoreactive B cells, and CD1d-deficiency in lupus mice exacerbates autoantibody production and enhances Ab response to a self peptide but not to a foreign peptide. Importantly, iNKTs fail to inhibit autoantibody production by CD1d-deficient B cells. Thus, iNKTs inhibit autoreactive B cells in a contact- and CD1d-dependent manner, but activate non-autoreactive B cells via cytokines. Such ability of iNKTs to suppress autoantibody production, without causing global suppression of B cells, has important implications for the development of iNKT-based therapy for autoimmune diseases.
doi:10.4049/jimmunol.1002373
PMCID: PMC3039137  PMID: 21209282
Rodent; B cells; T cells; Autoimmunity; Systemic lupus erythematosus
15.  Anti‐cyclic citrullinated peptide positivity in non‐rheumatoid arthritis disease samples: citrulline‐dependent or not? 
Annals of the Rheumatic Diseases  2006;66(4):511-516.
Background
Antibodies directed against citrullinated proteins (eg anti‐cyclic citrullinated peptide (CCP)) have excellent diagnostic and good prognostic potential for rheumatoid arthritis. Type 1 autoimmune hepatitis (AIH‐1) is a chronic liver disease characterised by a variety of serum autoantibodies. Recently, in a large group of patients with AIH‐1 without clear rheumatoid arthritis overlap, a relatively high percentage (9%) of anti‐CCP2 positivity was scored.
Objectives
To characterise the citrulline‐dependence of the observed anti‐CCP2 positivity in AIH‐1 sera as well as in other groups of patients without rheumatoid arthritis (mainly rheumatic diseases).
Methods
Serum samples of 57 patients with AIH‐1 and 66 patients without rheumatoid arthritis, most of them reported as anti‐CCP positive, were tested for citrulline‐specific reactivity with a second generation anti‐CCP kit, with the citrullinated and the corresponding non‐citrullinated (arginine‐containing) antigen. A subset of AIH‐1 sera was also tested with a CCP1 ELISA (and arginine control).
Results
The anti‐CCP2 reactivity of most non‐rheumatoid arthritis rheumatic diseases samples (87–93%) was citrulline‐specific, whereas a relatively high percentage of AIH‐1 samples (42–50%) turned out to be reactive in a citrulline‐independent manner. The use of citrullinated and non‐citrullinated CCP1 peptides confirmed a high occurrence of citrulline‐independent reactivity in AIH‐1 samples.
Conclusions
In rheumatoid arthritis and most non‐rheumatoid arthritis rheumatologic disease sera, anti‐CCP positivity is citrulline‐dependent. However in some patients, particularly patients with AIH‐1, citrulline‐independent reactivity in the anti‐CCP2 test can occur. A positive CCP test in a non‐rheumatic disease (eg liver disease) should therefore be interpreted with care, and preferably followed by a control ELISA with a non‐citrullinated antigen.
doi:10.1136/ard.2006.058933
PMCID: PMC1856034  PMID: 16984940
16.  Rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value, associations with radiological progression rate, and extra-articular manifestations 
Annals of the Rheumatic Diseases  2004;63(12):1587-1593.
Background: Autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein antibodies can be detected in rheumatoid arthritis (RA) sera.
Objective: To determine the diagnostic values of RF, anticitrullinated protein antibodies, and the shared epitope (SE), and their associations with radiological progression rates and extra-articular manifestations.
Methods: Population 1 consisted of sera from 315 patients, consecutively sent for detection of anticitrullinated protein antibodies, of which 264 were used to determine the sensitivity and specificity of RF and of antibodies against three synthetic citrullinated peptides: peptide A (pepA), peptide B (pepB), and CCP2. Population 2 consisted of sera from 180 longstanding RA patients and was used to determine associations of RA associated antibodies and the SE with radiological progression rates and extra-articular manifestations. Antibodies to pepA and pepB were detected by line immunoassay, and antibodies to CCP2 by ELISA. HLA Class II typing was performed by LiPA.
Results: In population 1, we defined adapted cut offs corresponding to a specificity of ⩾98.5%. This yielded the following sensitivities: RF 12.8%; anti-pepA antibodies 63.6%; anti-pepB antibodies 54.2%; and anti-CCP2 antibodies 73.7%. In population 2, significant differences in radiological progression rates were found between positive and negative patients for different RA antibodies and the SE. RF, but not anticitrullinated protein antibodies or the SE, were more frequent in patients with extra-articular manifestations.
Conclusion: A valid comparison of RA associated antibodies shows superior sensitivity of the anticitrullinated protein antibodies compared with RF. The presence of RA associated antibodies and the SE are indicative for poorer radiological outcome, and presence of extra-articular manifestations is associated with RF but not with anticitrullinated protein antibodies.
doi:10.1136/ard.2003.017574
PMCID: PMC1754859  PMID: 15547083
17.  Differential Genetic Associations for Systemic Lupus Erythematosus Based on Anti–dsDNA Autoantibody Production 
PLoS Genetics  2011;7(3):e1001323.
Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti–dsDNA autoantibody production, a SLE–related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti–dsDNA autoantibody positive (anti–dsDNA +, n = 811) and anti–dsDNA autoantibody negative (anti–dsDNA –, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti–dsDNA + SLE. Far fewer and weaker associations were observed for anti–dsDNA – SLE. For example, rs7574865 in STAT4 had an OR for anti–dsDNA + SLE of 1.77 (95% CI 1.57–1.99, p = 2.0E-20) compared to an OR for anti–dsDNA – SLE of 1.26 (95% CI 1.12–1.41, p = 2.4E-04), with pheterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti–dsDNA + SLE and were not associated with anti–dsDNA – SLE. In conclusion, we identified differential genetic associations with SLE based on anti–dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti–dsDNA – SLE.
Author Summary
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can involve virtually any organ system. SLE patients produce antibodies that bind to their own cells and proteins (autoantibodies) which can cause irreversible organ damage. One particular SLE–related autoantibody directed at double-stranded DNA (anti–dsDNA) is associated with kidney involvement and more severe disease. Previous genome-wide association studies (GWAS) in SLE have studied SLE itself, not particular SLE manifestations. Therefore, we conducted this GWAS of anti–dsDNA autoantibody production to identify genetic associations with this clinically important autoantibody. We found that many previously identified SLE–associated genes are more strongly associated with anti–dsDNA autoantibody production than SLE itself, and they may be more accurately described as autoantibody propensity genes. No strong genetic associations were observed for SLE patients who do not produce anti–dsDNA autoantibodies, suggesting that other factors may have more influence in developing this type of SLE. Further investigation of these autoantibody propensity genes may lead to greater insight into the causes of autoantibody production and organ damage in SLE.
doi:10.1371/journal.pgen.1001323
PMCID: PMC3048371  PMID: 21408207
18.  Evaluation of Anti-Mutated Citrullinated Vimentin Antibodies, Anti-Cyclic Citrullinated Peptide Antibodies and Rheumatoid Factor in Omani Patients with Rheumatoid Arthritis 
Rheumatoid factor (RF) is currently used in the diagnosis of rheumatoid arthritis (RA). The discovery of anticitrullinated protein autoantibodies has led to the development of various new tests, such as anti-cyclic citrullinated peptide (anti-CCP) antibodies, and anti-mutated citrullinated vimentin (anti-MCV) antibodies, to diagnose RA. The aims of this study were to determine the sensitivity and specificity of anti-MCV antibodies in comparison with anti-CCP antibodies and RF in Omani Arab patients with RA and compare our findings with published values from different ethnic groups. The sensitivity of anti-MCV antibodies was 72% with 87% specificity. For anti-CCP antibodies the sensitivity was 52% and the specificity was 97%. The sensitivity of RF was 57% with 94% specificity. Anti-CCP antibodies have higher diagnostic specificity and positive predictive value than RF and anti-MCV antibodies. Anti-MCV antibodies have the highest sensitivity when compared to anti-CCP antibodies and RF. Anti-MCV antibodies do not appear to be very useful in the diagnosis of RA. However, long-term study is required to find out whether anti-MCV antibodies can be used as predictive test for incidence of RA.
doi:10.1155/2012/285854
PMCID: PMC3426236  PMID: 22934118
19.  Autoantibodies in rheumatoid arthritis: rheumatoid factors and anticitrullinated protein antibodies 
Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease, characterized by chronic, erosive polyarthritis and by the presence of various autoantibodies in serum and synovial fluid. Since rheumatoid factor (RF) was first described, a number of other autoantibodies have been discovered in RA patients. The autoantigens recognized by these autoantibodies include cartilage components, chaperones, enzymes, nuclear proteins and citrullinated proteins. However, the clinical significances and pathogenic roles of these antibodies are largely unknown except for RF and anticitrullinated protein antibodies (ACPAs), whose clinical usefulness has been acknowledged due to their acceptable sensitivities and specificities, and prognostic values. This review presents and discusses the current state of the art regarding RF and ACPA in RA.
doi:10.1093/qjmed/hcp165
PMCID: PMC2825384  PMID: 19926660
20.  Association of Autoimmunity to Peptidyl Arginine Deiminase Type 4 With Genotype and Disease Severity in Rheumatoid Arthritis 
Arthritis and rheumatism  2008;58(7):1958-1967.
Objective
Protein citrullination is an important posttranslational modification recognized by rheumatoid arthritis (RA)–specific autoantibodies. One of the citrullinating enzymes, peptidyl arginine deiminase type 4 (PAD-4), is genetically associated with development of RA in some populations, although the mechanism(s) mediating this effect are not yet clear. There have been descriptions of anti–PAD-4 autoantibodies in different rheumatic diseases. This study was undertaken to investigate whether anti–PAD-4 antibodies are specific to RA, are associated with disease phenotype or severity, and whether PAD-4 polymorphisms influence the anti–PAD-4 autoantibody response.
Methods
Sera from patients with established RA, patients with other rheumatic diseases, and healthy adults were assayed for anti–PAD-4 autoantibodies by immunoprecipitation of in vitro–translated PAD-4. The epitope(s) recognized by PAD-4 autoantibodies were mapped using various PAD-4 truncations. PAD-4 genotyping was performed on RA patients with the TaqMan assay. Joint erosions were scored from hand and foot radiographs using the Sharp/van der Heijde method.
Results
PAD-4 autoantibodies were found in 36–42% of RA patients, and were very infrequent in controls. Recognition by anti–PAD-4 autoantibodies required the 119 N-terminal amino acids, which encompass the 3 nonsynonymous polymorphisms associated with disease susceptibility. Strikingly, the anti–PAD-4 immune response was associated with the RA susceptibility haplotype of PADI4. Anti–PAD-4 antibodies were associated with more severe joint destruction in RA.
Conclusion
Our findings indicate that anti–PAD-4 antibodies are specific markers of RA, independently associated with more severe disease, suggesting that an anti–PAD-4 immune response may be involved in pathways of joint damage in this disease. Polymorphisms in the PADI4 gene influence the immune response to the PAD-4 protein, potentially contributing to disease propagation.
doi:10.1002/art.23596
PMCID: PMC2692635  PMID: 18576335
21.  Defining the genetic origins of three rheumatoid synovium-derived IgG rheumatoid factors. 
Journal of Clinical Investigation  1994;93(6):2545-2553.
A major diagnostic marker in most rheumatoid arthritis (RA) patients is the rheumatoid factor (RF), an autoantibody that binds to the Fc region of IgG. To delineate the Ig genes and the underlying mechanism for RF production in RA patients, we applied a systematic approach to define the genetic origins of three IgG RFs derived from the synovial fluid of two RA patients. The results show that two of three IgG RF have substantial numbers of somatic mutations in their variable (V) regions, ranging from 13 to 23 mutations over a stretch of 291-313 nucleotides, resulting in a frequency of 4.4-7.8%. However, one IgG RF has only one mutation in each V region. This result indicates that an IgG RF may arise from a germline gene by very few mutations. The mutations occur mainly in the complementarity-determining regions (CDRs), and the mutations in the CDRs often lead to amino acid substitutions. Five of the six corresponding germline V genes have been found to encode either natural autoantibodies or autoantibodies in other autoimmune disorders; and three of the six V genes have been found in fetal liver. Taken together with other results, the data show that (a) several potentially pathogenic RFs in RA patients arise from natural autoantibodies, and (b) only a few mutations are required to convert the natural autoantibodies to IgG RFs.
PMCID: PMC294479  PMID: 8200991
22.  Anti-Interferon Autoantibodies in Autoimmune Polyendocrinopathy Syndrome Type 1 
PLoS Medicine  2006;3(7):e289.
Background
The autoimmune regulator (AIRE) gene influences thymic self-tolerance induction. In autoimmune polyendocrinopathy syndrome type 1 (APS1; OMIM 240300), recessive AIRE mutations lead to autoimmunity targetting endocrine and other epithelial tissues, although chronic candidiasis usually appears first. Autoimmunity and chronic candidiasis can associate with thymomas as well. Patients with these tumours frequently also have high titre immunoglobulin G autoantibodies neutralising type I interferon (IFN)–α and IFN-ω, which are secreted signalling proteins of the cytokine superfamily involved in both innate and adaptive immunity.
Methods and Findings
We tested for serum autoantibodies to type I IFNs and other immunoregulatory cytokines using specific binding and neutralisation assays. Unexpectedly, in 60/60 Finnish and 16/16 Norwegian APS1 patients with both AIRE alleles mutated, we found high titre neutralising immunoglobulin G autoantibodies to most IFN-α subtypes and especially IFN-ω (60% homologous to IFN-α)—mostly in the earliest samples. We found lower titres against IFN-β (30% homologous to IFN-α) in 23% of patients; two-thirds of these (from Finland only) also had low titres against the distantly related “type III IFN” (IFN-λ1; alias interleukin-29). However, autoantibodies to the unrelated type II IFN, IFN-γ, and other immunoregulatory cytokines, such as interleukin-10 and interleukin-12, were much rarer and did not neutralise.
Neutralising titres against type I IFNs averaged even higher in patients with APS1 than in patients with thymomas. Anti–type I IFN autoantibodies preceded overt candidiasis (and several of the autoimmune disorders) in the informative patients, and persisted for decades thereafter. They were undetectable in unaffected heterozygous relatives of APS1 probands (except for low titres against IFN-λ1), in APS2 patients, and in isolated cases of the endocrine diseases most typical of APS1, so they appear to be APS1-specific.
Looking for potentially autoimmunising cell types, we found numerous IFN-α+ antigen-presenting cells—plus strong evidence of local IFN secretion—in the normal thymic medulla (where AIRE expression is strongest), and also in normal germinal centres, where it could perpetuate these autoantibody responses once initiated. IFN-α2 and IFN-α8 transcripts were also more abundant in antigen-presenting cells cultured from an APS1 patient's blood than from age-matched healthy controls.
Conclusions
These apparently spontaneous autoantibody responses to IFNs, particularly IFN-α and IFN-ω, segregate like a recessive trait; their high “penetrance” is especially remarkable for such a variable condition. Their apparent restriction to APS1 patients implies practical value in the clinic, e.g., in diagnosing unusual or prodromal AIRE-mutant patients with only single components of APS1, and possibly in prognosis if they prove to predict its onset. These autoantibody responses also raise numerous questions, e.g., about the rarity of other infections in APS1. Moreover, there must also be clues to autoimmunising mechanisms/cell types in the hierarchy of preferences for IFN-ω, IFN-α8, IFN-α2, and IFN-β and IFN-λ1.
Almost all of nearly 100 APS1 patients studied made large amounts of auto-antibodies that blocked the function of IFN-α and IFN-ω. The antibodies appeared early during development of the disease and may play a role in its etiology.
Editors' Summary
Background.
The human body is under constant attack by viruses, bacteria, fungi, and parasites, but the immune system usually prevents these pathogens from causing disease. To be effective, the immune system has to respond rapidly to foreign antigens (bits of protein specific to pathogens) while ignoring self-antigens. If tolerance to self-antigens breaks down, autoimmunity develops, often causing disease. There are many common autoimmune diseases—type I diabetes and multiple sclerosis, for example—but because these involve defects in many genes as well as environmental factors, the details of how autoimmunity develops remain unclear. Autoimmune polyendocrinopathy syndrome type 1 (APS1), however, is caused by defects in a single gene. Patients with this rare disease characteristically have defects (or mutations) in both copies of a gene called AIRE (for autoimmune regulator). In normal people, the protein product of this gene helps to establish tolerance to a subset of self-antigens. People carrying AIRE mutations make an autoimmune response against some of their own tissues, typically the endocrine (hormone-producing) tissues that control body metabolism. A major component of this autoimmune response are “autoantibodies” (antibodies are immune molecules that normally recognize and attack foreign substances, whereas autoantibodies are directed against the body's own molecules).
Why Was This Study Done?
For a diagnosis of APS1, a patient must have at least two of the following symptoms: recurrent, localized yeast infections (usually the first symptom of the disease to appear in early childhood), hypoparathyroidism (failure of the gland that controls calcium levels in the body), and Addison disease (failure of the steroid-producing adrenal glands, which help the body respond to stress). The researchers who did this study had previously noticed that these yeast infections and autoimmunity (usually against muscle) can also occur in patients with tumors of the thymus (thymomas). The thymus is the organ that generates immune cells called T cells. Generation of the T cell repertoire in the thymus involves selection of those T cells that recognize only foreign substances. T cells that can react against self-antigens are eliminated, and the AIRE gene is thought to be involved in this “education process.” Like those with APS1, patients with thymomas make autoantibodies not only against target organs (especially muscle in their case), but also against interferon alpha (IFN-α) and interferon omega (IFN-ω), two secreted immune regulators. The researchers wanted to know if patients with APS1 also make autoantibodies against interferons, because this could provide insights into how autoimmunity develops in APS1 and other autoimmune diseases.
What Did the Researchers Do and Find?
The researchers tested blood from nearly 100 APS1 patients for antibodies to IFN-α, IFN-ω, and other immunoregulatory cytokines. They found that almost all patients made large amounts of antibodies that blocked the function of IFN-α and IFN-ω; some also made lower amounts of antibodies against two related interferons, but none made blocking antibodies against unrelated interferons or other immune regulators. For many patients, serum samples were available at different times during their disease, which allowed the researchers to show that the antibodies appeared early in disease development, before the onset of yeast infections or damage to endocrine tissues, and their production continued for decades as the patient aged. Furthermore, only patients with APS1 made these antibodies—they were absent in patients with Addison disease alone, for example.
What Do These Findings Mean?
The discovery that autoantibodies to IFN-α and IFN-ω are made persistently in patients with APS1 suggests ways in which autoimmunity develops in these patients. These can now be investigated further both in patients and in animal models of the disease. The discovery also has practical implications. Measurement of these autoantibodies might help some APS1 patients by allowing earlier diagnosis—and prompter treatment—than in current practice. The levels of these autoantibodies might also help to predict the time course of APS1 in individual patients, although more studies will be needed to check this out. Finally, if future studies show that interferon autoantibodies are responsible for the patients' susceptibility to yeast infections (which seems plausible), treatment with IFN-γ, an interferon to which APS1 patients do not make autoantibodies, might provide an alternative way to deal with this problem.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030289.
• MedlinePlus pages on autoimmune diseases
• Online Mendelian Inheritance in Man page on APS1
• Links to patient information on APS1 from the Stanford Health Library
• Wikipedia page on autoendocrine polyendocrinopathy (note: Wikipedia is a free online encyclopedia that anyone can edit)
• Information on autoimmunity from the American Autoimmune Related Diseases Association
doi:10.1371/journal.pmed.0030289
PMCID: PMC1475653  PMID: 16784312
23.  Genome-Wide Association Analysis of Autoantibody Positivity in Type 1 Diabetes Cases 
PLoS Genetics  2011;7(8):e1002216.
The genetic basis of autoantibody production is largely unknown outside of associations located in the major histocompatibility complex (MHC) human leukocyte antigen (HLA) region. The aim of this study is the discovery of new genetic associations with autoantibody positivity using genome-wide association scan single nucleotide polymorphism (SNP) data in type 1 diabetes (T1D) patients with autoantibody measurements. We measured two anti-islet autoantibodies, glutamate decarboxylase (GADA, n = 2,506), insulinoma-associated antigen 2 (IA-2A, n = 2,498), antibodies to the autoimmune thyroid (Graves') disease (AITD) autoantigen thyroid peroxidase (TPOA, n = 8,300), and antibodies against gastric parietal cells (PCA, n = 4,328) that are associated with autoimmune gastritis. Two loci passed a stringent genome-wide significance level (p<10−10): 1q23/FCRL3 with IA-2A and 9q34/ABO with PCA. Eleven of 52 non-MHC T1D loci showed evidence of association with at least one autoantibody at a false discovery rate of 16%: 16p11/IL27-IA-2A, 2q24/IFIH1-IA-2A and PCA, 2q32/STAT4-TPOA, 10p15/IL2RA-GADA, 6q15/BACH2-TPOA, 21q22/UBASH3A-TPOA, 1p13/PTPN22-TPOA, 2q33/CTLA4-TPOA, 4q27/IL2/TPOA, 15q14/RASGRP1/TPOA, and 12q24/SH2B3-GADA and TPOA. Analysis of the TPOA-associated loci in 2,477 cases with Graves' disease identified two new AITD loci (BACH2 and UBASH3A).
Author Summary
Autoantibodies are important markers for autoimmune diseases such as type 1 diabetes and Graves' disease. However, little is known about the genetic factors that control their production. To improve our understanding of this genetic basis, we measured four autoantibodies in a collection of up to 8,300 type 1 diabetes cases plasma samples. We combined these measurements with genome-wide genotype data to conduct four independent genome-wide association studies. Two loci showed unequivocal evidence of autoantibody association: the FCRL3 locus and the ABO blood group locus. Variants in the FCRL3 gene have been previously associated with autoimmune diseases, but such associations have not been reported for ABO blood group genotypes. In addition, we found extensive overlap between type 1 diabetes and autoantibody loci, and these findings provide new information about the role of these risk variants. Lastly, we hypothesized that loci associated with thyroid autoantibodies are strong candidates for association with thyroid autoimmune disorders. We confirmed this hypothesis by genotyping these variants in an independent cohort of Graves' disease cases, and we found evidence for two new Graves' disease loci.
doi:10.1371/journal.pgen.1002216
PMCID: PMC3150451  PMID: 21829393
24.  Autoantibodies to Posttranslational Modifications in Rheumatoid Arthritis 
Mediators of Inflammation  2014;2014:492873.
Autoantibodies have been associated with human pathologies for a long time, particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known since the late 1930s to be associated with rheumatoid arthritis (RA). The discovery of anticitrullinated protein antibodies in the last century has changed this and other posttranslational modifications (PTM) relevant to RA have since been described. Such PTM introduce neoepitopes in proteins that can generate novel autoantibody specificities. The recent recognition of these novel specificities in RA provides a unique opportunity to understand human B-cell development in vivo. In this paper, we will review the three of the main classes of PTMs already associated with RA: citrullination, carbamylation, and oxidation. With the advancement of research methodologies it should be expected that other autoantibodies against PTM proteins could be discovered in patients with autoimmune diseases. Many of such autoantibodies may provide significant biomarker potential.
doi:10.1155/2014/492873
PMCID: PMC3981057  PMID: 24782594
25.  Major histocompatibility complex class II genes and systemic sclerosis. 
Annals of the Rheumatic Diseases  1991;50 (Suppl 4):862-865.
1. In no ethnic group is the overall association between systemic sclerosis and the MHC strong enough for direct clinical use. MHC associations do support the classification of the disease into limited cutaneous systemic sclerosis and diffuse cutaneous systemic sclerosis. 2. Indications are that associations between specific subsets of patients with systemic sclerosis and genetic markers will assume greater importance both diagnostically and prognostically. The group with lung fibrosis look prime candidates, for example. 3. Genetic markers are useful means of relating chemically induced systemic sclerosis like disorders with the classical disease. Vinyl chloride disease provides an example. 4. Evidence is emerging of strong associations between certain genetic markers and autoantibody production; a similar story has emerged in systemic lupus erythematosus. We believe that, eventually, genetic tests will be used to influence treatment in at least a subset of patients with systemic sclerosis but that a dramatic breakthrough will not be made until we know how the genetics of the disease relate to the primary biochemical disease characteristic--that is, the overproduction of collagen. In this respect it has been suggested that the 5' flanking DNA of dermal collagen genes is particularly susceptible to the action of Scl-70 (topoisomerase I). A problem is how to tie this and the other observations discussed above together. The association of autoantibodies with topoisomerase I provides a tentative link between the MHC and collagen gene expression. Although the role and reason for anti-Scl-70 in systemic sclerosis is unknown, humoral autoimmunity, at least in systemic lupus erythematosus, seems to be strongly dependent on specific HLA genes. With an understanding of the function of MHC products at the molecular level, HLA and disease associations can now be analysed on a mechanistic level. For insulin dependent diabetes mellitus it has been shown that the MHC determined susceptibility to the disease is conferred by neutral residues (Val, Ser, Ala), at position 57 of the DQ beta chain, while Asp at this position correlates with resistance. A similar phenomenon has been described in rheumatoid arthritis. Although DR4 in general is associated with rheumatoid arthritis, it is heterogeneous, but a subtype of DR4 which is characterised by positively charged residues at positions 70 and 71 of the beta chains is not found in patients with rheumatoid arthritis (Wordsworth B P et al, unpublished data). A similar approach applied to the study of systemic sclerosis is likely to be similarly rewarding. The precise subtyping of the class II genes and the characterisation of their associated haplotypes is therefore required for a complete understanding of the contribution of the MHC to the disease. Additional genes linked to the MHC must not be overlooked, and are relevant to associations of haplotypes with the disease. Of particular interest are the recent reports of a new class of proteins, which are determined by genes in the MHC and which are considered to play a part in the assembly of the antigen peptide/MHC molecule complex.
PMCID: PMC1033321  PMID: 1750798

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