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Objective: Antibodies to cyclic citrullinated peptides (anti-CCP) in rheumatoid arthritis (RA) can express the inherently autoreactive gene V H 4-34, detected using the rat monoclonal antibody 9G4. Patients with the polyarticular subtype of juvenile idiopathic arthritis (JIA) share some but not all of the features of adult patients with RA. This study was undertaken to compare serologic findings for rheumatoid factor (RF), anti-CCP, and 9G4-expressing anti-CCP in a large JIA cohort with a cohort of adult RA patients., Methods: Serum from 88 patients with polyarticular JIA, 29 patients with enthesitis-related arthritis, 38 patients with extended oligoarthritis, 31 adolescent controls, 35 patients with RA, and 30 adult controls were tested for RF, for IgG, IgA, and IgM anti-CCP, and for 9G4-expressing anti-CCP by enzyme-linked immunosorbent assay. Total serum 9G4-positive IgM was also measured., Results: Of 65 patients with RF-negative polyarticular JIA, 4 (6.2%) were IgG anti-CCP positive. Sera from 20 of 23 patients with RF-positive polyarticular JIA (87.0%), 24 of 35 patients with RA (68.6%), and 1 patient with extended oligoarthritis contained IgG anti-CCP. IgA and IgM anti-CCP levels were lower in the adolescent group (P < 0.01). Levels of 9G4-expressing anti-CCP were higher in patients with RF-positive polyarticular JIA than in those with RF-negative polyarticular JIA (P < 0.0001). Median levels of 9G4-expressing anti-CCP in patients with RF-positive polyarticular JIA and those with RA did not differ. Expression of 9G4 on serum total IgM was greater in patients with RF-positive polyarticular JIA than other adolescent groups (P < 0.01), but similar to adult RF-positive RA., Conclusion: In healthy individuals, 9G4-positive B cells comprise 5-10% of the peripheral blood pool but serum immunoglobulins utilizing V H 4-34 are disproportionately low. The idiotope recognized by 9G4 was detected on anti-CCP antibodies in >80% of patients with RF-positive polyarticular JIA. V H 4-34 usage by anti-CCP in both JIA and RA patients suggest elicitation of these autoantibodies through shared pathogenic B cell selection processes., (© 2017, American College of Rheumatology.)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterised by multiple symptoms including fatigue, headaches and cognitive impairment, which have a significantly adverse effect on the normal functioning and well-being of the individual. These symptoms are often triggered or worsened following physical or mental exertion. ME/CFS has long been thought of as having a significant immunological component, but reports describing changes in immune function are often inconsistent between study groups. Although the wide range of physical, neurocognitive and autonomic symptoms reported have seriously hampered attempts to understand pathophysiological pathways, investment in biomedical research in ME/CFS is finally increasing with a number of novel and promising investigations being published. The onset of ME/CFS may often be linked to (viral) infections which would be consistent with a variety of alterations in natural killer (NK) cell function as described by a number of different groups. Consistency in cytokine data has been lacking so far, although recently more sophisticated approaches have led to more robust data from large patient cohorts. New hope has also been given to sufferers with the possibility that therapies that deplete B cells can result in clinical improvement. To understand the pathogenic mechanism in this complex condition, it is important to consider repeated analysis in different cohorts. In this review, we will discuss the potential of different components of the immune system to be involved in the pathogenesis of ME/CFS., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Objective: Increased cardiovascular risk has been associated with high levels of serum antibodies to citrullinated proteins (ACPA) in patients with rheumatoid arthritis (RA). Citrullination is part of many chronic inflammatory processes and we therefore investigated whether ACPA might be associated with coronary artery disease, in the absence of RA., Methods: To maximize the potential predictive value of this retrospective study we included sera from a cohort of 3052 healthy male individuals, subsequently followed for the development of coronary artery disease, and documented for other disease risk factors. With each case event (myocardial infarction; n = 144), 2 matched controls were assigned., Results: We found 10.4% of cases were ACPA positive compared to 3.8% of controls (odds ratio (95% CI) = 3.26 (1.36-7.80), p = 0.008), remaining significant after adjustment for classical risk factors including smoking and CRP (4.23 (1.22-14.61) p = 0.02)., Conclusion: The genesis and fine specificity of ACPA in patients with atherosclerosis, in the absence of Rheumatoid arthritis, may prove worthy of further investigation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objective: To assess the relationships between serum B lymphocyte stimulator (BLyS) levels, autoantibody profile and clinical response in patients with systemic lupus erythematosus (SLE) following rituximab-based B cell depletion therapy (BCDT)., Methods: A total of 25 patients with active refractory SLE were followed for >or=1 year following BCDT. Disease activity was assessed using the British Isles Lupus Assessment Group (BILAG) system, and serum levels of BLyS and autoantibodies to dsDNA and extractable nuclear antigens (ENA) measured by ELISA. Serum immunoglobulins and anti-dsDNA antibodies were assessed for expression of the 9G4 idiotope (indicating VH4-34 germline gene origin)., Results: Following BCDT, all patients depleted in the peripheral blood and improved clinically for >or=3 months. Pre-BCDT BLyS levels were quantifiable (median 1.9 ng/ml) in 18/25 patients and rose in most patients at 3 months post-BCDT (median 4.15 ng/ml). Nine patients, all with quantifiable pre-BCDT serum BLyS, experienced a disease flare within 1 year. This group of patients was more likely to harbour anti-Ro/SSA antibodies (odds ratio 1.76; p = 0.06) with higher serum levels (p = 0.0027; Mann-Whitney U test). Serum levels of anti-ribonucleoprotein (RNP)/Sm were also higher in this group (p<0.05). Expression of VH4-34 by serum immunoglobulins and anti-dsDNA antibodies had no predictive value for the length of clinical response., Conclusions: Patients with SLE with an expanded autoantibody profile and raised BLyS levels at baseline had shorter clinical responses to BCDT. This may reflect a greater propensity to, and degree of, epitope spreading in such patients and suggests that treatment regimens beyond BCDT may be necessary to induce long-lasting clinical remissions in these individuals.

Objective: Autoantibody production in patients with systemic lupus erythematosus (SLE) is associated with abnormalities of B cell function and phenotype. Clinical responses to B cell depletion therapy (BCDT), based on rituximab, are encouraging. Therefore, we undertook this study to investigate the effect of BCDT on antibody profiles., Methods: Serial sera from 16 patients with active, refractory SLE were assayed for antinucleosome antibodies, anti-double-stranded DNA (anti-dsDNA), anti-extractable nuclear antigen, anti-tetanus toxoid, and antibodies to pneumococcal capsular polysaccharide for at least 1 year following BCDT. Anti-dsDNA antibodies derived from the V(H)4.34 immunoglobulin germ line gene (9G4+) were also measured., Results: All patients achieved peripheral B cell depletion and improved clinically for at least 3 months. Antinucleosome and anti-dsDNA antibodies decreased to a mean +/- SD of 64 +/- 37% and 38 +/- 33% of baseline values, respectively, by 6-8 months post-BCDT. Levels of other autoantibodies and antimicrobial antibodies were generally unchanged. In the 9 of 16 patients who were still well at 1 year, anti-dsDNA antibodies fell to 42 +/- 36% of baseline values at 6-8 months and to 37 +/- 33% at 10-14 months. In patients who had disease flares within 1 year of BCDT, levels of these antibodies decreased to 60 +/- 40% and 83 +/- 93% of baseline values at 6-8 months and at 10-14 months, respectively. Circulating anti-dsDNA antibodies were positive for 9G4 expression in 4 of 6 patients tested, and flares in 2 of these patients were accompanied by rises in 9G4+ anti-dsDNA antibodies., Conclusion: These observations suggest that B cell clones committed to producing antinucleosome and anti-dsDNA antibodies, including the V(H)4.34 subpopulation of anti-dsDNA antibodies, have a relatively rapid turnover compared with B cell clones producing other antibodies. There was also a trend toward a greater and more sustained decrease in anti-dsDNA antibodies in patients with clinical benefit lasting >1 year.

Occular involvement is infrequent and blindness rare in Churg-Strauss syndrome. We describe a patient with Churg-Strauss syndrome who presented with blindness. This was associated with the appearance of circulating autoantibodies to myeloperoxidase.

Objective: Although antimyeloperoxidase (MPO) antibodies provide a sensitive serological test for vasculitis, their significance in sera from a small proportion (about 10%) of patients with autoimmune rheumatic disease is controversial. Our aim was to determine the incidence of anti-MPO antibodies in sera from patients with systemic sclerosis (SSc) and their relation to renal disease., Methods: Thirty-eight patients had limited cutaneous SSc (lSSc) and 43 diffuse cutaneous SSc, and within each group, 24 and 27 patients, respectively, had renal impairment (defined as stable creatinine clearance less than 60 ml/min). Six patients previously had had a scleroderma renal crisis. After screening for antineutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence, the levels of IgM and IgG anti-MPO antibodies in 8 patients with SSc was determined by ELISA, with human MPO as antigen., Results: Sera from 2 patients, both with lSSc and renal impairment, were perinuclear p-ANCA positive and had significant levels of circulating IgM and IgG anti-MPO antibodies. In one patient, anti-MPO antibodies appeared in the serum only after D-penicillamine was introduced, continued to rise after withdrawal of the drug, and fell only after immunosuppressive therapy. Renal biopsy confirmed vasculitis. The 2nd patient died of unrelated disease before further investigations could be performed., Conclusion: We suggest that circulating anti-MPO antibodies are not a feature of SSc per se and, if found, may indicate the presence of an unrelated pathology, such as idiopathic or drug induced vasculitis.

Circulating antibodies to myeloperoxidase (MPO) are associated primarily with pauci-immune glomerulonephritis and systemic vasculitis. Anti-MPO antibodies belong to a group of autoantibodies, anti-neutrophil cytoplasmic antibodies, that may play a pathogenic role in vasculitis. We have generated a human monoclonal anti-MPO antibody (E3-MPO) using peripheral blood lymphocytes from a patient with microscopic polyarteritis. Variable region gene analysis of E3-MPO showed that the VH region had 90% homology with the germ line gene VH4-21. E3-MPO was also shown to carry the 9G4 idiotope, which so far has been associated only with human antibodies that utilize the VH4-21 gene. The 9G4 idiotope was also expressed on anti-MPO antibodies in sera from the donor patient and from 4/7 additional patients with active, untreated vasculitis. The nucleotide sequences of both the variable heavy and light chains of E3-MPO showed evidence of an antigen-driven response.

Background/aims: Antineutrophil cytoplasmic antibodies have been reported to occur more frequently in healthy first-degree relatives of patients with ulcerative colitis than in healthy controls. The aim of this study was to determine their prevalence in families in which more than one member was affected with inflammatory bowel disease., Methods: With use of an indirect immunofluorescence method, 168 affected members and 197 unaffected first-degree relatives in 56 such families were studied., Results: Antibodies were detected in 46% of patients with ulcerative colitis without a positive family history and in 44% of those within families with several affected members. There was no evidence of clustering of antibodies in particular families. Within families in which both ulcerative colitis and Crohn's disease coexisted, antibodies were detected primarily in patients with ulcerative colitis. Antibodies were seldom present (3%) in those patients with Crohn's disease, whether they were familial or nonfamilial cases. Similarly, antibodies were seldom present (3%) in controls or the unaffected first-degree relatives of patients with inflammatory bowel disease., Conclusions: Antineutrophil cytoplasmic antibodies are associated with ulcerative colitis, and their presence is not increased in the first-degree relatives of patients with ulcerative colitis. They are more likely to be a consequence of the disease than a subclinical genetic disease marker.

Circulating antibodies to myeloperoxidase (MPO) have been described in a variety of vasculitic syndromes, drug-induced SLE and drug-induced nephritis. We have examined the autoantibody profile in acute sera from patients with antineutrophil cytoplasmic antibody-positive vasculitis (n = 8), drug-induced nephritis (n = 4), drug-induced lupus (n = 7), SLE (n = 27) and nephritis-associated with SLE (n = 17). Significant binding to purified MPO in ELISA was given by all sera from patients with vasculitis and drug-induced nephritis but ANA sought by indirect immunofluorescence on HEp-2 cells were not detected. Both anti-MPO and ANA were found in sera from patients with drug-induced lupus. Sera from patients with SLE or SLE nephritis did not contain high titres of anti-MPO antibodies but invariably contained ANA. Anti-MPO antibodies of both IgG and IgM classes were present in all sera from patients with drug-induced disease. Although the number of samples tested was small, sera from patients with drug-induced nephritis showed significantly greater median % binding of IgM to MPO compared with drug-induced SLE. Binding to MPO by IgG in these sera was not significantly different. These findings suggest that the mechanism of interaction between hydralazine and the immune system in the two drug-induced autoimmune diseases studied may contribute to their distinct clinical features.

Objectives: To determine the prevalence and clinical associations of autoantibodies to myeloperoxidase (MPO) in an unselected series of well-characterised outpatients with rheumatoid arthritis (RA) and to compare the distribution of IgG subclasses of anti-MPO antibodies in these patients with that in patients with systemic vasculitis., Patients and Methods: A study was made of 97 patients with RA, who have been seen regularly in this department for up to 20 years, and 29 patients with anti-neutrophil cytoplasmic antibody (ANCA) positive systemic vasculitis. Anti-MPO antibodies were detected using a direct-binding enzyme-linked immunosorbent assay (ELISA) with MPO from human granulocytes as antigen. The IgG subclass of anti-MPO antibodies was determined by ELISA using isotype specific monoclonal antibodies., Results: Anti-MPO antibodies were detected in 12% of patients with RA. Six sera contained IgG anti-MPO antibodies only, 1 IgM only and 5 antibodies of both classes. In the patients with RA the predominant subclasses were IgG1 and IgG3: only 2 sera contained detectable IgG4 antibodies. This was in contrast to patients with vasculitis, in whom most sera contained IgG1, IgG3 and IgG4 anti-MPO antibodies. Anti-MPO antibodies in sera from both patient groups bound only to the native protein. None of the patients studied with RA had evidence of vasculitis affecting the nerves or kidney: three patients (1 positive for anti-MPO antibodies and 2 negative) had cutaneous vasculitis. In the patients with RA, positivity for anti-MPO antibodies was associated with nodules and number of active joints. Three patients with anti-MPO antibodies, and none without, had pulmonary fibrosis., Conclusions: Twelve per cent of a group of unselected outpatients with RA, but without evidence of major systemic vasculitis, had anti-MPO antibodies in their serum. Positivity for anti-MPO antibodies was more common in patients with nodular disease and lung involvement but not in patients with cutaneous vasculitis. IgG4 sub-class anti-MPO antibodies were present in 90% of sera from patients with ANCA-positive vasculitis and only 2/11 (18%) of anti-MPO antibody containing sera from patients with RA.

Antineutrophil cytoplasmic antibodies (ANCAs) are found in sera from patients with vasculitis and less commonly in sera from other autoimmune diseases. ANCAs provide the only specific diagnostic test for vasculitis and are also useful in monitoring disease activity. The indirect immunofluorescence test remains the best screening test for ANCAs.

Antibodies to endothelial cells may be involved in the pathogenesis of vasculitic disorders. In view of recent evidence implicating intestinal vascular injury in the pathogenesis of inflammatory bowel disease, we have sought anti-endothelial cell antibodies in affected patients, examined their relationship to vascular injury, and tested their ability to mediate endothelial cell cytotoxicity in vitro. Anti-endothelial cell antibody levels were elevated in ulcerative colitis (P < 0.0001) and Crohn's disease (P < 0.05) compared with healthy controls. In ulcerative colitis, anti-endothelial cell antibody levels were related to disease activity and correlated with circulating levels of von Willebrand factor (r = 0.58, P < 0.01), a marker of vascular injury. Anti-endothelial cell antibodies, however, were not directly cytotoxic to endothelial cells in vitro. These data indicate, for the first time, an association between anti-endothelial cell antibody levels and vascular injury in vivo and suggest that they may be important in the pathogenesis of inflammatory bowel disease, particularly ulcerative colitis.

Anti-neutrophil cytoplasmic antibodies (ANCA) in sera from patients with clinically proven vasculitis have been described as reacting with proteins present in the granules of human neutrophils. We have studied sera from 59 ANCA positive patients to further characterize the antibody response. In addition to the antigens previously identified in the vasculitic syndromes (myeloperoxidase and serine proteinase 3) the majority of these sera contained antibodies that reacted with a cytosolic extract of neutrophils on Western blots. Nearly 40% of these sera had antibodies directed against a cytosolic protein(s) of molecular mass 48 kD. This protein was purified from neutrophil cytosol by ammonium sulphate fractionation, anion exchange and reverse phase chromatography. Amino acid sequence analysis of a proteolytic fragment of this protein identified it as alpha enolase. The anti-enolase antibodies only recognized the alpha isoform and were present in sera giving either a pANCA or cANCA staining pattern by indirect immunofluorescence. Antibodies to alpha enolase were also found in sera from patients with systemic lupus erythematosus, particularly those with renal disease. We conclude that the antibody response in ANCA positive vasculitis is not restricted to neutrophil granule proteins.

Two mouse-human heterohybridomas secreting human antibodies to myeloperoxidase (MPO) were derived from the peripheral blood of a patient who developed microscopic polyarteritis as the result of long-term treatment with hydralazine. Forty-five immunoglobulin-secreting lines were obtained from the fusion of patient lymphocytes with the CB-F7 heteromyeloma cell line. Of these, two antibodies, one IgG and one IgM, bound to myeloperoxidase in solid phase ELISA and gave a perinuclear staining pattern on ethanol-fixed human neutrophil cytospin preparations. The staining patterns were similar to those seen with serum from the patient. Antigen-inhibition studies revealed that the affinity of the IgG monoclonal antibody was 28 times higher (k = 1.4 x 10(-7)) than the IgM antibody (k = 5 x 10(-5)). Cross-inhibition studies further suggested that the two monoclonal antibodies recognized the same epitope on MPO. Of the other secreting cell lines, none produced antibody which reacted with the panel of autoantigens used for testing. Neither mononuclear antibody reacted with this panel indicating that they were not simply polyreactive natural autoantibodies. These are the first human monoclonal antibodies to native myeloperoxidase to be reported.

Anti-neutrophil antibodies have been shown in sera from patients with a variety of inflammatory diseases. Those reacting with components of neutrophil cytoplasm are associated with systemic vasculitis. Both nuclear and perinuclear staining patterns on human neutrophils have been reported using sera from patients with inflammatory bowel disease. We have evaluated the reactivity against human neutrophils of sera from 100 patients with inflammatory bowel disease, 14 disease controls, and 20 normal volunteers. Altogether 27/50 (54%) sera from patients with ulcerative colitis contained antibodies that reacted with cytospun ethanol fixed neutrophils compared with 5/50 (10%) from Crohn's disease (p less than 0.001) and 0/34 control sera (p less than 0.001). All seven sera from patients with proctitis alone were negative (p less than 0.01). There was no correlation between presence or titre of anti-neutrophil antibodies and either disease activity or treatment. Positive sera gave three different staining patterns on human neutrophils. The predominant pattern was perinuclear (17/32); 12 sera gave a cytoplasmic and three a homogeneous nuclear staining pattern. None of the patients or the controls had antibodies to myeloperoxidase, elastase, or serine proteinase 3, all of which are recognised by anti-neutrophil cytoplasmic antibodies. Only 2/27 sera positive by indirect immunofluorescence reacted with an extract of neutrophil primary granules. In conclusion, anti-neutrophil antibodies occur more commonly in ulcerative colitis than in Crohn's disease or control subjects and the anti-neutrophil antibodies found in inflammatory bowel disease are different from those associated with vasculitis.

The clinical expression of disease in patients with conditions in which autoimmunity is thought to contribute to the pathogenesis of disease is the result of an unfortunate combination of predisposing and environmental factors. The presence of autoantibodies showing a variety of antigen specificities in sera from many of these patients has been closely correlated with particular spectra of organ involvement or tissue destruction. Their precise role in the disease process is as yet unclear. Sera from patients with paraproteinaemia also often contain autoantibodies to a variety of cell components, although symptoms of autoimmune disease are rarely found in this group of individuals. In this study of 42 sera from patients with paraproteinaemia we have confirmed the presence of autoantibodies in 33% (13/42) of samples. Amongst the autoantibodies detected were those to human neutrophils (3), U1RNP (8) and cardiolipin (4). In five sera, the immunoglobulin class of autoantibody did not correlate with that of the monoclonal band. This study extends previous reports of the repertoire of autoantibodies present in sera from patients with paraproteinaemia.

Abstract: B-cell depletion therapy (BCDT) based on rituximab (RTX) induces clinical remission in a majority of seropositive patients with Rheumatoid arthritis (RA). However, all patients eventually relapse. The aim of this study was to determine whether dynamic changes in combinations of serological measures of B-cell activation were associated over up to three cycles of BCDT. We included only RA patients who gave an adequate clinical response, as measured by DAS28. Twenty three patients were studied over 1 cycle, 21 over 2, and 15 over 3 cycles of BCDT. Serum analytes including isotypes of Rheumatoid factors (RhF) and anti-citrullinated protein/peptide antibodies (ACPA), B-cell activating factor (BAFF), serum free light chains (SFLC), soluble CD23 (sCD23), antibodies to tetanus toxoid (TT) and to pneumococcal capsular polysaccharide (PCP) were measured by ELISA at 4 key points in each cycle, namely: Baseline (pre-RTX in each cycle); when B-cell depleted (CD19+B-cells < 5/μl); at B-cell return (CD19+B-cells ≥ 5/μl); and at clinical relapse (ΔDAS28 > 1.2). SFLC were used as a measure of plasmablast activity. As sCD23 is cleaved from naïve B-cells coincident with attaining CD27 expression, levels were used as a novel measure of maturation of B-cells to CD27+. The most consistent changes between baseline and B-cell depletion within all 3 cycles were in SFLC, sCD23 and IgM-RhF which fell and in BAFF levels which rose. After 3 complete cycles of BCDT, both IgM autoantibodies and IgG-CCP had decreased, BAFF levels were higher (all p < 0.05); other analytes remained unchanged compared with baseline. Dynamic changes in λSFLC, sCD23, IgM-RhF and BAFF were also consistently associated with relapse in patients with longer clinical responses after B-cell return. Incremental rises in sCD23 levels in cycles 2 and 3 were correlated with time to relapse. Repopulation of the periphery after BCDT is initiated by naïve B-cells and precedes relapse. Our study showed that differentiation into plasmablasts, attended by sCD23 and SFLC production and IgM-RhF specificity may be required to precipitate relapse in patients experiencing longer responses after RTX. These studies also provide novel information related to the resumption of autoimmune responses and their association with B-cell kinetics following BCDT. [Copyright &y& Elsevier]

Objective: To compare the levels of the 9G4 idiotope (9G4 Id) in systemic lupus erythematosus (SLE) patients with a detailed disease activity index, the British Isles Lupus Assessment Group (BILAG) index, and serological parameters of disease activity by ds DNA antibody levels and serum C3 concentrations.Methods: In a cross sectional analysis serum samples from 190 patients with SLE were studied and a further 55 serial bleeds from 14 patients. An enzyme linked immunosorbent assay was used to measure the 9G4 Id, and anti dsDNA and antimyeloperoxidase (MPO) antibodies. The C3 levels were measured by laser nephelometer.Results: Seventy six of 190 (40%) of the patients tested had raised 9G4 Id levels. In the cross sectional study 9G4 Id levels were found to correlate with disease activity in the BILAG cardiovascular/respiratory renal, and haematological systems and with global BILAG score (p < 0.01). In the serial bleeds 9G4 Id levels correlated with anti-dsDNA antibody and C3 levels, but not with anti-MPO antibodies. No correlations were found with treatment. In six cases the 9G4 Id levels correlated well with global BILAG scores and dsDNA antibody levels. In four cases the BILAG global and 9G4 Id levels alone correlated well.Conclusions: Raised levels of the 9G4 Id are present in a substantial proportion of serum samples from patients with lupus, correlate with various aspects of disease activity in SLE. The Id is detectable on anti-dsDNA antibodies, though it must also be present on other immunoglobulins whose specificities remain unknown. [ABSTRACT FROM AUTHOR]