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.     

Immunoglobulin A anti‐phospholipid antibodies in Swedish cases of systemic lupus erythematosus: associations with disease phenotypes,vascular events and damage accrual

Immunoglobulin (Ig) G‐ and IgM‐class anti‐cardiolipin antibodies (aCL) and lupus anti‐coagulant (LA) are included in the 1997 update of the American College of Rheumatology (ACR‐97) systemic lupus erythematosus (SLE) criteria. Despite limited evidence, IgA‐aCL and IgA anti‐β₂‐glycoprotein‐I (anti‐β₂GPI) were included in the 2012 Systemic Lupus International Collaborating Clinics criteria. The present study aimed to evaluate IgG‐/IgA‐/IgM‐aCL and anti‐β₂GPI occurrence in relation to disease phenotype, smoking habits, pharmacotherapy, anti‐phospholipid syndrome (APS) and organ damage among 526 Swedish SLE patients meeting ACR‐97. Patients with rheumatoid arthritis (n = 100), primary Sjögren’s syndrome (n = 50) and blood donors (n = 507) served as controls. Anti‐phospholipid antibodies (aPL) were analysed by fluoroenzyme‐immunoassays detecting aCL/anti‐β₂GPI. Seventy‐six (14%) SLE cases fulfilled the Sydney APS‐criteria, and ≥ 1 aCL/anti‐β₂GPI isotype (IgG/IgA/IgM) occurred in 138 SLE patients (26%). Forty‐five (9%) of the SLE cases had IgA‐aCL, 20 of whom (4%) lacked IgG‐/IgM‐aCL. Seventy‐four (14%) tested positive for IgA anti‐β₂GPI, 34 (6%) being seronegative regarding IgG/IgM anti‐β₂GPI. Six (1%) had APS manifestations but were seropositive regarding IgA‐aCL and/or IgA anti‐β₂GPI in the absence of IgG/IgM‐aPL and LA. Positive LA and IgG‐aPL tests were associated with most APS‐related events and organ damage. Exclusive IgA anti‐β₂GPI occurrence associated inversely with Caucasian ethnicity [odds ratio (OR) = 0·21, 95% confidence interval (CI) = 0·06–0·72) and photosensitivity (OR = 0·19, 95% CI = 0·05–0·72). Nephritis, smoking, LA‐positivity and statin/corticosteroid‐medication associated strongly with organ damage, whereas hydroxychloroquine‐medication was protective. In conclusion, IgA‐aPL is not rare in SLE (16%) and IgA‐aPL analysis may have additional value among SLE cases with suspected APS testing negative for other isotypes of aPL and LA.     

Label‐free detection of immune complexes with myeloid cells

The aim of this study was to provide proof‐of‐concept for quantitative and qualitative label‐free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA‐specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme‐linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen‐specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen‐specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2‐based serological positivity and European League Against Rheumatism (EULAR) criteria‐based clinical RA diagnosis. Immunoglobulin‐triggered binding of monocytoid cells can be monitored using a label‐free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti‐citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc‐receptor‐expressing cells is also affected by post‐translational modification of the immunoglobulins.     

Clinical significance of autoantibodies recognizing Sjögren's syndrome A (SSA), SSB, calpastatin and alpha-fodrin in primary Sjögren's syndrome

The aim of our study was (i) to compare the clinical and biological characteristics of 148 (137 women, 11 men) primary Sjögren's syndrome (pSS) patients at diagnosis as a function of their sex and (ii) to assess the prognostic value of anti‐calpastatin and anti‐alpha‐fodrin autoantibodies. In addition, the presence of anti‐nuclear antibodies (ANA), anti‐52‐ and 60‐kDa Sjögren's syndrome A (SSA), anti‐Sjögren's syndrome B (SSB), anti‐cyclic citrullinated peptide (CCP) antibodies and rheumatoid factors (RF) of IgA, IgG and IgM isotypes was sought in sera collected at pSS onset. Raynaud's syndrome, significantly more frequent in women, was the only systemic manifestation of pSS whose frequency differed significantly as a function of the patient's sex (P = 0·02). ANA (P = 0·001) and anti‐60‐kDa SSA autoantibodies (P = 0·03) were significantly more common in women, while men never synthesized detectable levels of anti‐SSB, anti‐calpastatin or IgG anti‐alpha‐fodrin autoantibodies. In addition, anti‐CCP autoantibodies were found in low percentages of pSS patients (4% F/18% M). The absence of autoantibodies does not exclude the diagnosis of pSS in men that will be based mainly on the anatomopathological findings of a minor salivary gland biopsy. Positivity of anti‐60‐kDa SSA, anti‐SSB, anti‐calpastatin, IgA and IgG anti‐alpha‐fodrin antibodies is not associated with pSS clinical and biological severity.     

Diverse IgG subclass responses to adeno‐associated virus infection and vector administration

Humoral immune responses occur following exposure to Adeno‐associated virus (AAV) or AAV vectors. Many studies characterized antibody responses to AAV, but human IgG subclass responses to AAV have not been previously described. In this study, IgG subclass responses were examined in serum samples of normal human subjects exposed to wild‐type AAV, subjects injected intramuscularly with AAV vectors and subjects injected intravascularly with AAV vectors. A diversity of IgG subclass responses to AAV capsid were found in different subjects. IgG1 was found to be the dominant response. IgG2, IgG3, and IgG4 responses were also observed in most normal human subjects; IgG2 and IgG3 each represented the major fraction of total anti‐AAV capsid IgG in a subset of normal donors. Subjects exposed to AAV vectors showed IgG responses to AAV capsid of all four IgG subclasses. IgG responses to AAV capsid in clinical trial subjects were inversely proportional to the level of pre‐existing anti‐AAV antibody and independent of the vector dose. The high levels of anti‐AAV capsid IgG1 can mask differences in IgG2, IgG3, and IgG4 responses that were observed in this study. Analysis of IgG subclass distribution of anti‐AAV capsid antibodies indicates a complex, non‐uniform pattern of responses to this viral antigen. J. Med. Virol. 81:65–74, 2009. © 2008 Wiley‐Liss, Inc.     

A peptide mimic blocks the cross‐reaction of anti‐DNA antibodies with glomerular antigens

Anti‐DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross‐reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti‐DNA antibodies to self‐antigens is isotype‐dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti‐DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9‐11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti‐DNA IgG isotypes to glomerular antigens. The PL9‐11 panel of IgG anti‐DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12‐mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme‐linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or ‘ALW’) which binds to all PL9‐11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9‐11 anti‐DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti‐DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti‐DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti‐DNA antibody binding to renal tissue.     

Subclass Distribution of IgA and IgG Antibodies Against Clq in Patients with Rheumatic Diseases

To obtain insight into the immunoregulatory mechanisms in patients with different rheumatic diseases, the occurrence and the subclass distribution of IgA and IgG antibodies against Clq (anti-ClqAb) was determined. In patients with systemic lupus erythaematosus (SLE) the highest frequency of increased serum levels of IgG anti-ClqAb were found, whereas IgA anti-ClqAb were predominantly present in patients with ankylosing spondylitis (AS) and patients with rheumatoid arthritis complicated by vasculitis (RV). In all the IgA anti-ClqAb positive AS and RV patients the antibody reactivity involved the IgAl subclass while the IgA2 subclass was found in 47% of the patients. Further characterization of the IgA anti-Clq binding activity in sera of AS patients revealed that both subclasses of IgA anti-ClqAb were predominantly polymeric; the binding of both IgA subclasses with solid phase CI q was inhibitable by aggregated fluid phase Clq; we found no delectable interference of rheumatoid factor in the test system for the measurement of IgA anti-ClqAb. In patients with SLE the IgG anti-ClqAb reactivity was mainly of the IgG2 and IgG3 subclass, whereas in the same patients the IgG anti-tetanus toxoid response was not restricted to these subclasses.
The predominance of IgG2 and IgG3 subclass of anti-ClqAb in sera of SLE patients, suggests a skewing of the anti-ClqAb response. The observation that the IgA anti-ClqAb of both subclasses is predominantly polymeric in nature and the notion that polymeric IgA is associated with activation of inflammation cascades, suggests that IgA anti-ClqAb may contribute to tissue damage.     

Subclass distribution of IgG and IgA antibody response to Pseudomonas pseudoalcaligenes in humans exposed to infected metal-working fluid

The subclass distribution of the IgG and IgA antibody response in serum was studied in humans exposed to aerosolized metal-working fluid containing Pseudomonas pseudoalcaligenes. This species was consistently found in concentrations of 10(8) bacteria per milliliter of metal-working fluid during 1 year of observation. No increased frequency of respiratory infections or discomfort was related to the exposure to the infected fluid. The antibody response to the bacterium consisted predominantly of IgG1 and IgG2 antibodies. IgG2 antibodies dominated the antibody response to the lipopolysaccharide of the bacterium. IgA1 and IgA2 antibodies were also found. Smokers had significantly reduced antibody levels of all subclasses compared with nonsmokers. The antibody levels in smokers did not differ from levels of the unexposed control group. Analyses of the total serum immunoglobulin concentrations with respect to subclasses revealed that the total IgG2 levels were also significantly reduced in smokers. In nonsmokers, the age of the individuals influenced the antibody levels of the IgG1, IgG2, IgA1, and IgA2 subclasses, the levels decreasing with increasing age. For smokers, the correlation between age and antibody levels was only obvious for IgG2 antibodies. Decreased IgG2 antibody levels in the smokers were also accompanied by decreased FEV1 values (p less than 0.01). Subclass analysis of the antibody response to P. pseudoalcaligenes demonstrated that the subclass pattern for the whole bacterium differed from the pattern of the major cell wall component, the lipopolysaccharide. The significance of qualitative and quantitative differences in the subclass antibody response is discussed.     

Differential decline in Leishmania membrane antigen-specific immunoglobulin G (IgG), IgM, IgE, and IgG subclass antibodies in Indian kala-azar patients after chemotherapy

Pathogenesis in kala-azar is associated with depressed cellular immunity and significant elevation of antileishmanial antibodies. Since these antibodies are present even after cure, analysis of the parasite-specific isotypes and immunoglobulin G (IgG) subclasses in kala-azar patients may shed new light on the immune responses during progression and resolution of infection. Using leishmanial membrane antigenic extracts, we investigated the relative levels of specific IgG, IgM, IgA, IgE, and IgG subclasses in Indian kala-azar patient sera during disease, drug resistance, and cure. Acute-phase sera showed strong stimulation of IgG, followed by IgE and IgM and lastly by IgA antibodies. IgG subclass analysis revealed expression of all of the subclasses, with a predominance of IgG1 during disease. Following sodium stibogluconate (SAG) resistance, the levels of IgG, IgM, IgE, and IgG4 remained constant, while there was a decrease in the titers of IgG2 and IgG3. In contrast, a significant (2.2-fold) increase in IgG1 was observed in these individuals. Cure, in both SAG-responsive and unresponsive patients, correlated with a decline in the levels of IgG, IgM, IgE, and all of the IgG subclasses. The stimulation of IgG1 and the persistence, most importantly, of IgE and IgG4 following drug resistance, along with a decline in IgE, IgG4, and IgG1 with cure, demonstrate the potential of these isotypes as possible markers for monitoring effective treatment in kala-azar.     

IgG subclasses and DNA detection of HHV‐6 and HHV‐7 in healthy individuals

Human herpesvirus 6 (HHV‐6) and 7 (HHV‐7) are common opportunistic agents in immunocompromised hosts, although infection with HHV‐6 and HHV‐7 can also be observed in immunocompetent hosts. Despite similar biology and epidemiology, this study evaluated differences in the IgG subclass distribution associated with HHV‐6 and HHV‐7 in seropositive, healthy persons. The identified subclasses were also compared with the detection of HHV‐6 and HHV‐7 DNA. For these assays, sera, plasma, and saliva samples were obtained from 40 healthy blood donors in Argentina who were seropositive for both HHV‐6 and HHV‐7. HHV‐6 and HHV‐7 DNA were detected in saliva and plasma samples using nested PCR, and specific IgG subclasses were determined using immunofluorescent assays of sera samples. HHV‐7 DNA was detected in 90% of all plasma samples and in 100% of saliva samples. In contrast, HHV‐6 DNA was not detected in any of the plasma samples, and it was detected in only 6 of 40 saliva samples. Determination of IgG subclass distributions showed that HHV‐6 was restricted to IgG1, whereas HHV‐7 IgG subclasses included two groups, one restricted only to IgG1 and the other to IgG1 and IgG3. These results demonstrate the differences between HHV‐6 and HHV‐7 DNA range detection in saliva and plasma samples, as well as the IgG subclass patterns for each virus type, in healthy persons in Argentina. J. Med. Virol. 82:1679–1683, 2010. 2010 Wiley‐Liss, Inc.     

Anti‐Citrullinated Peptide Antibodies in Sudanese Patients with Leishmania donovani Infection Exhibit Reactivity not Dependent on Citrullination

African patients with Leishmania donovani infections have signs of strong systemic inflammation and high levels of circulating immune complexes (IC) and rheumatoid factor (RF), all serologic markers of rheumatic disease. As inflammation in general is associated with citrullination, we sought to investigate ACPA responses in Sudanese Leishmania patients. Serum samples were collected from Sudanese patients with visceral leishmaniasis (VL) and post‐kala‐azar dermal leishmaniasis (PKDL) as well as from ACPA‐positive Sudanese rheumatoid arthritis patients and compared to healthy Sudanese controls. Levels of circulating C1q‐binding IC and anticyclic citrullinated peptide 2(CCP2) were investigated using ELISA, and RF was measured with nephelometry. C1q adsorption was carried out to investigate anti‐CCP2 content in IC. Citrulline specificity was evaluated with control plates with cyclic arginine‐containing control peptides. Leishmania‐infected patients had elevated levels of RF and circulating IC but also a significant increase in anti‐CCP2 (12%) as compared to healthy controls. Anti‐CCP2‐positive Leishmania patients displayed lower anti‐CCP2 levels than Sudanese patients with rheumatoid arthritis (RA), and anti‐CCP2 levels in Leishmania patients showed a continuum not resembling the dichotomous pattern seen in patients with RA. Whereas the anti‐CCP reactivity of Sudanese RA sera was strictly citrulline dependent, anti‐CCP2‐positive Leishmania sera reacted equally well with ELISA plates containing arginine control peptides. There was a strong correlation between anti‐CCP2 and circulating IC among the Leishmania patients, but IC depletion only marginally diminished anti‐CCP2 levels. Our findings stress the importance to interpret a positive CCP test carefully when evaluated in non‐rheumatic conditions associated with macrophage activation.     

Galectin‐3 is Persistently Increased in Early Rheumatoid Arthritis (RA) and Associates with Anti‐CCP Seropositivity and MRI Bone Lesions,While Early Fibrosis Markers Correlate with Disease Activity

Galectin‐3 has been suggested as a pro‐inflammatory mediator in animal arthritis and rheumatoid arthritis (RA). We aimed to study the serum level of galectin‐3 in patients with newly diagnosed RA and associations with disease profile, Magnetic resonance imaging (MRI) findings and seromarkers of synovial matrix inflammation. One hundred and sixty DMARD naïve patients newly diagnosed with RA were included (CIMESTRA study). Clinical, serological and imaging data were recorded before treatment and at 6 weeks, 3 and 12 months. Galectin‐3 and hyaluronan (HYA) were measured by ELISA (R&D and Corgenix, USA), and the N‐terminal propeptide of type III collagen (PIIINP) by radioimmunoassay (Orion Diagnostica, Finland). One hundred and nineteen, 87 and 60 blood donors served as controls for galectin‐3, HYA and PIIINP, respectively. Baseline galectin‐3 was significantly elevated in anti‐CCP positive (4.2 μg/l IQR [3.6;6.1]) patients as compared with anti‐CCP negatives (4.0 μg/l [2.6;4.9], P = 0.05) and controls (3.8 μg/l [3.0;4.8], P < 0.01). During treatment, galectin‐3 remained elevated, but increased transiently with peak values at 6 weeks. Galectin‐3 correlated with baseline smoking, anti‐CCP, and with MRI erosion score after 1 year of follow‐up. HYA and PIIINP were elevated (P < 0.001) irrespective of anti‐CCP status and correlated positively with synovitis assessed clinically and by MRI. HYA and PIIINP did not correlate with galectin‐3. These observations indicate that HYA and PIIINP mainly reflect expansive synovitis proliferation while galectin‐3 is more closely linked to autoimmunity, smoking and joint destructive processes.     

Lipopolysaccharide- and cholera toxin-specific subclass distribution of B-cell responses in cholera.

The immunoglobulin subclass responses to homologous lipopolysaccharide (LPS) and to cholera toxin (CT) in adult patients infected with Vibrio cholerae O1 and V. cholerae O139 were studied. LPS-specific antibody-secreting cells (ASC) of both the immunoglobulin A1 (IgA1) and IgA2 subclasses were seen, with the IgA1 ASC response predominating in both V. cholerae O1- and O139-infected patients. For antibodies in plasma, by day 11 after onset of disease, all V. cholerae O1- infected patients responded to homologous LPS with the IgA1 subclass (P = 0.001), whereas fewer (68%) responded with the IgA2 subclass (P = 0.007). About 89% of V. cholerae O139-infected patients responded with the IgA1 subclass (P = 0.003), and only 21% responded with the IgA2 subclass (not significant [NS]). Both groups of cholera patients showed significant increases in LPS-specific IgG1, IgG2, and IgG3 antibodies in plasma. In feces, the response to homologous LPS occurred in both groups of patients with the IgA1 and IgA2 subclasses, with 55 to 67% of patients showing a positive response. V. cholerae O1- and O139-infected patients showed CT-specific ASC responses of the different IgG and IgA subclasses in the circulation, and the pattern followed the order IgG1 > IgA1 > IgG2 > IgA2, with low levels of IgG3 and IgG4 ASC. Plasma anti-CT antibody responses in all subclasses were seen by day 11 after onset of disease. Although there were no increases in CT-specific ASC of the IgG3 (NS) and IgG4 (NS) subtypes, there were significant increases of these two subclasses in plasma (P 相似文献   

IgA‐ and SIgA anti‐PR3 antibodies in serum versus organ involvement and disease activity in PR3‐ANCA‐associated vasculitis

Circulating immunoglobulin (Ig)A class anti‐neutrophil cytoplasm antibodies (ANCA) directed against proteinase 3 (PR3) have been reported in ANCA‐associated vasculitis (AAV) with mucosal involvement. However, secretory IgA (SIgA) PR3‐ANCA has not been reported previously. In this study we compared serum levels of SIgA PR3‐ANCA and IgA PR3‐ANCA with IgG PR3‐ANCA in relation to disease characteristics. Among 73 patients with AAV and PR3‐ANCA at diagnosis, 84% tested positive for IgG PR3‐ANCA, 47% for IgA‐ANCA and 36% for SIgA PR3‐ANCA at the time of sampling for the present study. IgA and IgG PR3‐ANCA were represented similarly among patients with different organ manifestations, i.e. upper airway, lung or kidney at time of sampling. However, SIgA PR3‐ANCA was significantly less represented among patients with upper airway involvement. During active disease, the proportions of IgA PR3‐ANCA and SIgA PR3‐ANCA‐positive patients were significantly higher compared to inactive disease. Eight patients were sampled prospectively during 24 months from onset of active disease. In these patients, IgA PR3‐ANCA and SIgA PR3‐ANCA turned negative more often after remission induction compared to IgG PR3‐ANCA. Our findings suggest that serum IgA PR3‐ANCA and SIgA PR3‐ANCA are related more closely to disease activity in AAV compared to IgG PR3‐ANCA. Further studies are required to reveal if this has implications for disease activity monitoring. The mean number of PR3‐ANCA isotypes increased along with disease activity, suggesting a global B cell activation during active disease.     

Bacteroides-specific IgG and IgA subclass antibody-secreting cells isolated from chronically inflamed gingival tissues

The emergence of cells that produce IgG and IgA subclass antibodies to Bacteroides gingivalis (Porphyromonas gingivalis) fimbriae and lipopolysaccharide (LPS) antigens was examined in mononuclear cells isolated from inflamed gingiva of different stages (slight, moderate or advanced) of adult periodontitis (AP). Antigen-specific IgM, IgG (including IgG1, IgG2, IgG3 and IgG4) and IgA (including IgA1 and IgA2) producing cells were enumerated by the ELISPOT assay and were compared with total Ig-producing cells of each isotype or subclass. In advanced AP, the B. gingivalis fimbriae-specific IgG- and IgA-secreting cells represented 5% of total IgG- or IgA-secreting cells, while those from the moderate stage comprised approximately 1% of these two isotypes. Cells producing antibody specific for B. gingivalis LPS were observed at frequencies of 0.1% and 0.4% for IgG and IgA cells, respectively in the advanced stage. When IgG subclasses were analysed in moderate AP, the anti-fimbriae subclass responses were largely IgG1 (60%), followed by IgG2 (20%), IgG3 (10%) and IgG4 (10%). Fimbriae-specific IgG subclass responses were elevated in the advanced stage of AP, and IgG4 (40%) and IgG1 (30%) were dominant, followed by IgG3 (20%) and IgG2 (10%). IgA1 cells predominated in both the moderate and advanced stages, however a relative increase in IgA2 cells occurred in advanced AP. Mononuclear cells isolated from gingiva of AP patients did not contain cells producing antibody to antigens such as Escherichia coli K235 LPS, cholera toxin or the hapten dinitrophenyl coupled to bovine serum albumin. These results show that local IgG and IgA subclass responses occur to a protein antigen of a major periodontal disease (PD)-associated pathogen, B. gingivalis, and the increase in IgG4 and IgA2 responses may be associated with host protection.     

Immunoglobulin G (IgG) and IgA subclass pattern of human antibodies to Shigella flexneri and Salmonella serogroup B and D lipopolysaccharide O antigens

The subclass distribution of human serum antibodies to the O-antigenic lipopolysaccharides of Salmonella serogroups B and D and to Shigella flexneri serotypes 1b, 2a, and 4a lipopolysaccharide antigens were analyzed in an enzyme-linked immunosorbent assay with monoclonal antibodies to the immunoglobulin subclasses. The patients had culture-verified Salmonella (17 Swedes) or Shigella flexneri (23 Vietnamese; 11 children and 12 adults) infections. Consecutive samples drawn during 1 year postinfection were investigated. Antibodies to the Salmonella antigens were mainly of the immunoglobulin G1 (IgG1), IgA1, and IgA2 subclasses. For the Salmonella serogroup B O polysaccharide, the IgA1 and IgA2 subclasses had peak values earlier than (6/9) or coinciding with the IgG1 (3/9) peak value. Furthermore, the IgA2 response to Salmonella serogroup B was positively correlated to the duration of the carrier state (P less than 0.001); the corresponding IgA1 response was less well correlated but was still significant (P less than 0.02). In the case of the Shigella flexneri O polysaccharide, specific antibodies appeared mainly in the IgG1 and IgA1 subclasses. Some IgG2 was also found, surprisingly even in very young patients. No subclass shift with time within the immunoglobulin classes was noted in any of the groups.     

Anti-Beta 2-glycoprotein I antibody isotype and IgG subclass in antiphospholipid syndrome patients

Beta 2-glycoprotein I (beta2-GPI) is an antigenic target recognised by antiphospholipid antibodies found in association with the antiphospholipid syndrome (APS). In this study, the prevalence of Immunoglobulin M (IgM) and IgA anti-beta2-GPI antibodies was examined in APS patients and compared with IgG antibodies. In addition the value of measuring antibody isotypes and IgG subclass was investigated in the laboratory diagnosis of APS. A solid phase enzyme linked immunosorbent assay was established to measure IgG, IgM and IgA and IgG subclass antibodies to beta2-GPI in patients with APS and a variety of other thrombotic and non-thrombotic disorders. Raised levels of IgM anti-beta2-GPI antibodies were observed in 65% of patients with APS, 21% with systemic lupus erythematosus (SLE), 23% with rheumatoid factor, 4% with stroke, 5% carotid artery stenosis (CAS), 17% with a biological false positive serology for syphilis, 43% with infectious mononucleosis (IM) and 27% with human immunodeficiency virus (HIV). The median value for IgM antibodies to beta2-GPI for all these groups ranged from 2 to 7 arbitrary units (AU). Elevated levels of IgA antibodies to beta2-GPI were found in patients with APS (47%), SLE (13%), rheumatoid factor (26%), CAS (48%), stroke (25%), VDRL false positive serology for syphilis (33%), IM (47%) and HIV (7%). The median value of IgA antibodies to beta2-GPI in all of these groups ranged from 2 to 4 AU. Conversely the median value for IgG anti-beta2-GPI in APS patients was 112 AU compared to 1-4 AU in the other conditions examined. The presence of IgM and IgA antibodies to beta2-GPI was much less specific and sensitive for APS than IgG, with raised levels of these isotypes seen in a variety of thrombotic and non-thrombotic disorders. Elevated levels of IgG1, IgG2, IgG3 and IgG4 antibodies to beta2-GPI were detected in APS patients. While all four IgG anti-beta2-GPI antibody subclasses were represented in APS patients there appeared to be a significant overall skewing towards to the IgG2 subclass.     

Relationship between natural and infection‐induced antibodies in systemic autoimmune diseases (SAD): SLE,SSc and RA

Infection or vaccine‐induced T cell‐dependent immune response and the subsequent high‐affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease‐specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine‐induced and disease‐related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme‐linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti‐CS) and F4 fragment (anti‐F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n = 92), rheumatoid arthritis (n = 73) and systemic sclerosis (n = 157) disease groups. Anti‐measles IgG and anti‐dsDNA IgG/IgM autoantibodies were measured using commercial and in‐house indirect ELISA tests. In all SAD groups the anti‐measles IgG‐seropositive cases showed significantly higher anti‐CS IgG titers (P = 0·011). In anti‐dsDNA IgG‐positive SLE patients, we detected significantly higher levels of anti‐CS and anti‐F4 IgG nAAbs (P = 0·001 and < 0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti‐CS and anti‐F4 nAAbs in anti‐dsDNA IgM‐positive SLE patients (P = 0·002 and 0·016, respectively). The association between IgG isotypes of pathogen‐ or autoimmune disease‐related antibodies and the IgG nAAbs may underscore the immune response‐inducible nature of the diseases investigated. The relationship between protective anti‐dsDNA IgM and the IgM isotype of anti‐F4 and anti‐CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.     

Immunoglobulin class and IgG subclass distribution of anticardiolipin antibodies in patients with systemic lupus erythematosus and associated disorders.

The class and subclass distribution of an antibody response may give insight into the stimulating mechanism and likely effector functions. IgA, IgG and IgM anticardiolipin antibodies (aCL) were quantified in a consecutive series of 200 samples sent to an autoimmune serology laboratory to determine the relationships between aCL responses of each of these antibody classes and, in particular, whether there was any utility in the measurement of IgA aCL. Positive results for one of the three aCL isotypes were found in 105 samples (53%), and in 41 samples IgA aCL was detected (21%). However, amongst these unselected samples, little additional information was obtained by measurement of IgA aCL, which was found in conjunction with IgM or IgG aCL in all but five samples, and in these the isolated elevation of IgA aCL was only slight, and showed no disease specificity. The levels of each of the four IgG subclasses of aCL were measured in a subgroup of serum samples from 28 patients with autoimmune disease and from 29 patients with syphilis. Amongst the SLE patients IgG1 and IgG3 aCL were the predominant IgG subclasses, consistent with an antigen-driven, T cell-dependent antibody response. However, a subgroup of eight of the autoimmune subjects had predominant elevation of IgG2 aCL, possibly implying a role for T cell-independent antibody production to cardiolipin. Amongst the syphilis patients IgG1 and IgG3 aCL were also the predominant subclasses of aCL but IgG4 aCL were also detected in the majority of subjects, consistent with prolonged antigenic stimulation.     

Comparative analysis of autoantibodies targeting peptidylarginine deiminase type 4, mutated citrullinated vimentin and cyclic citrullinated peptides in rheumatoid arthritis: associations with cytokine profiles,clinical and genetic features

Antibodies against cyclic citrullinated peptides (anti‐CCP) are widely used for diagnosis of rheumatoid arthritis (RA). We performed a comparative analysis of antibodies targeting the citrullinating enzyme peptidylarginine deiminase type 4 (anti‐PAD4) and mutated citrullinated vimentin (anti‐MCV) with anti‐CCP autoantibodies in RA patients and examined their relationships with clinical parameters, cytokine profiles and the PADI4 gene. Autoantibodies were examined by enzyme‐linked immunosorbent assay (ELISA) in sera of 170 RA patients and 103 controls. Cytokine profiles were measured using a multiplex system. PADI4 polymorphisms (89G > A, 90T > C and 92G > C) were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Anti‐PAD4, anti‐MCV and anti‐CCP autoantibodies were detected in 24, 61 and 74% of RA patients, respectively. Positive correlations were observed between anti‐PAD4 and disease duration; anti‐CCP and erythrocyte sedimentation rate (ESR); anti‐MCV and ESR and C‐reactive protein. Anti‐MCV antibodies were associated with high disease activity score 28 (DAS‐28) in early RA. Concentrations of T helper type 1 (Th1) [tumour necrosis factor (TNF)‐α, interleukin (IL)‐12, IL‐2, IL‐1β], Th2 (IL‐4, IL‐6, IL‐10, IL‐13) and Th17 (IL‐17) cytokines were higher in RA than in controls. Th2 and, to a lesser extent, Th1‐related cytokines, showed positive correlations with anti‐MCV and anti‐CCP. The GTG haplotype in PADI4 was associated with anti‐CCP and anti‐MCV, but not anti‐PAD4 antibodies. In conclusion, anti‐PAD4 antibodies are detected mainly in established RA, which is in contrast to the early detection of antibodies against citrullinated peptide/proteins (ACPAs). Among autoantibodies, anti‐MCV appear to perform better as markers of disease activity. Furthermore, anti‐CCP and anti‐MCV are associated genetically with the citrullinating enzyme PAD4 and are related strongly to Th1 and Th2 cytokines, suggesting a feed‐forward loop between cytokines and ACPA production.