Lone N. Troelsen

Double role of mannose-binding lectin in relation to carotid intima-media thickness in patients with rheumatoid arthritis.

BACKGROUND Patients with rheumatoid arthritis (RA) have increased risk of atherosclerosis and cardiovascular disease (CVD) that cannot be explained by excess of traditional risk factors. Several studies indicate that mannose-binding lectin (MBL) may modify the development of atherosclerosis; both high and low serum levels of MBL are reported to be associated with CVD. Intima-media thickness of the common carotid artery (ccIMT) is a validated non-invasive anatomic measure of subclinical CVD. We examined the relation between ccIMT and MBL in 114 RA patients. METHODS In a cross-sectional study MBL2 genotypes and serum concentrations of MBL were assessed; ccIMT was determined by means of ultrasonography; traditional and RA related cardiovascular risk modifiers were measured. RESULTS The median ccIMT was 0.67 mm. The investigated MBL2 genotypes were not significantly associated with ccIMT. Using a general linear model, ccIMT was not linearly associated with serum MBL but was highly associated with the quadratic term of serum MBL (MBL(2)) (P=0.001) reflecting a U-shaped relation. MBL(2) was also significantly associated with ccIMT in a multivariable analysis adjusting for traditional and RA related cardiovascular risk modifiers (P=0.025). CONCLUSION In RA patients, a quadratic U-shaped relation between serum MBL and ccIMT was observed independently of the effects of traditional and RA related risk factors for CVD. These results provide further support to the notion that both high and low levels of MBL may be associated with CVD.

Mortality and Predictors of Mortality in Rheumatoid Arthritis - A Role for Mannose-binding Lectin?

Objective. Patients with rheumatoid arthritis (RA) have increased overall and cardiovascular mortality. Mannose-binding lectin (MBL) may play differentiated roles in the pathogenesis of RA. We had observed that high serum levels of MBL increased the risk of ischemic heart disease in patients with RA. In this followup study we describe the mortality in a cohort of 229 Danish patients with RA. We examine if previously reported factors and MBL influence the risk of overall death and death due to cardiovascular disease. Methods. Known predictors of RA mortality were assessed. MBL extended genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, YO/YO) were determined; MBL serum concentrations were measured. The vital status and causes of death were assessed in a prospective study. Results. The median followup was 10.3 years. The overall risk of death was 4% per year. Comparing mortality in the RA cohort with mortality in an age- and sex-matched cohort based on the general Danish population, we found significantly increased overall mortality [standardized mortality ratio (SMR) 1.5, 95% CI 1.2–1.9, and cardiovascular mortality (SMR 1.7, 95% CI 1.3–2.6)]. In multivariate analysis, significant predictors of overall death were extraarticular manifestations, positive rheumatoid factor, increased C-reactive protein (CRP), poor nutritional state, and serum MBL. Predictors of cardiovascular death were Health Assessment Questionnaire score, increased CRP, poor nutritional state, and the high-producing MBL genotype YA/YA. Conclusion. Both overall and cardiovascular mortality were increased in Danish patients with RA. In our cohort, states of high MBL production and several previously reported factors contributed significantly to this increased risk of overall death and cardiovascular death.

IgG glycosylation changes and MBL2 polymorphisms: associations with markers of systemic inflammation and joint destruction in rheumatoid arthritis.

Objective. To examine whether IgG glycosylation changes and MBL2 genotypes are associated with systemic inflammation and joint destruction in rheumatoid arthritis (RA). Methods. IgG N-glycan content was determined from serum in 118 patients with RA by high-throughput glycan analysis using normal-phase high-pressure liquid chromatography. MBL2 extended genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, YO/YO) were determined. Systemic inflammation was assessed by serum levels of C-reactive protein (CRP), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α). Joint destruction was assessed by total Sharp score (TSS) and alloplastic surgery records. Results. IgG hypogalactosylation was significantly correlated to IL-6 (Spearman’s rho = 0.32, p < 0.001), CRP (Spearman’s rho = 0.31, p < 0.001), TSS (Spearman’s rho = 0.25, p = 0.01), and alloplastic replacement of joints (Spearman’s rho = 0.18, p = 0.05). In multivariate analysis including age, CRP, anticitrullinated protein antibodies (ACPA), and other confounders, IgG hypogalactosylation was significantly associated with TSS (p = 0.014) and alloplastic joint replacement (OR 76.5, p = 0.041) in patients homozygous for the high expression MBL2 genotype YA/YA, but not in carriers of lower expression genotypes. Conclusion. Decreased galactosylation of IgG correlated to markers of inflammation, i.e., IL-6 and CRP. Only in patients homozygous for high expression of the MBL2 genotype YA/YA was IgG hypogalactosylation associated with markers of joint destruction. Our results suggest that inflammation-associated decreased galactosylation of IgG combined with high expression MBL2 genotypes are involved in the pathophysiology of RA.

Genetically determined serum levels of mannose-binding lectin correlate negatively with common carotid intima-media thickness in systemic lupus erythematosus.

Objective. Patients with systemic lupus erythematosus (SLE) have excess cardiovascular morbidity and mortality due to accelerated atherosclerosis that cannot be attributed to traditional cardiovascular risk factors alone. Variant alleles of the mannose-binding lectin gene (MBL2) causing low serum concentrations of functional mannose-binding lectin (MBL) are associated with SLE and development of atherosclerosis. Recent studies show that these variant alleles are associated with increased risk of arterial thrombosis and cardiovascular disease in patients with SLE. Intima-media thickness of the common carotid artery (ccIMT) is a validated noninvasive anatomic measure of subclinical atherosclerosis. In a cross-sectional study we examined the relation among ccIMT, MBL2 genotypes, and serum concentrations of MBL. Methods. The MBL2 extended genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, YO/YO) and serum concentrations of MBL were determined in 41 outpatients with SLE. ccIMT was measured by means of ultrasonography. Traditional and nontraditional cardiovascular risk modifiers were assessed and controlled for. Results. Using nonparametric Mann-Whitney tests we found a significant difference in ccIMT between low-expressing (XA/XA+YA/YO+XA/YO+YO/YO) and high-expressing (YA/YA+YA/XA) MBL2 genotypes (p = 0.034). The difference in ccIMT remained significant in multivariable analysis adjusting for traditional and nontraditional cardiovascular risk modifiers (p = 0.049). ccIMT was negatively correlated to serum concentrations of MBL (Spearman rho = −0.33, p = 0.037). This relation also remained significant in multivariable analysis (p = 0.042). Conclusion. In this group of SLE patients, MBL2 low-expressing genotypes and low serum levels of MBL were correlated with ccIMT, independent of the effects of traditional and nontraditional cardiovascular risk modifiers.

Isotypes of Epstein-Barr Virus Antibodies in Rheumatoid Arthritis: Association with Rheumatoid Factors and Citrulline-Dependent Antibodies

In order to study the humoral immune response against Epstein-Barr virus (EBV) in patients with rheumatoid arthritis (RA) and to compare it with the two major autoantibody types in RA, plasma samples from 77 RA patients, 28 patients with systemic lupus erythematosus (SLE), and 28 healthy controls (HCs) were investigated by enzyme-linked immunosorbent assays (ELISA). Increased percentages of positives and concentrations of IgG/IgA/IgM antibodies against the latent EBV nuclear antigen-1 (EBNA-1) were observed in RA patients compared to SLE patients and HCs. Increased concentrations and percentages of positives of IgG/IgA/IgM against the early lytic EBV antigen diffuse (EAD) were also found in RA patients compared to HCs but were highest in SLE patients. Furthermore, associations between the elevated EBNA-1 IgA and EBNA-1 IgM levels and the presence of IgM and IgA rheumatoid factors (RFs) and anti-citrullinated protein antibodies (ACPAs, IgG) and between elevated IgA concentrations against EAD and the presence of RFs and ACPAs in RA patients were found. Thus, RA patients had elevated antibodies of all isotypes characteristic of latent EBV infection (whereas SLE patients had elevated antibodies characteristic of lytic EBV infection). Notably, for IgM and IgA (but not IgG), these were associated with the presence of characteristic RA autoantibodies.

Melanoma Inhibitor of Apoptosis Protein (ML-IAP) Specific Cytotoxic T Lymphocytes Cross-React with an Epitope from the Auto-Antigen SS56

A large proportion of melanoma patients host a spontaneous T-cell response specifically against ML-IAP-derived peptides. In this study, we describe that some ML-IAP-specific cytotoxic T cells isolated from melanoma patients cross react with an epitope from the auto-antigen SS56. SS56 is a recently described target of autoantibody responses in Sjögrens syndrome (SS) as well as systemic lupus erythematosus (SLE). Here, we describe that SS56 is also an auto-antigen for T cells in SS and SLE. Hence, SS56-specific T cells could readily be detected in circulation and among the infiltrating cells of SLE skin lesions. SS56-specific T cells were able to lyse target cells presenting the peptide epitope on the surface. Notably, SS56-specific CD8 T cells isolated from an SS patient cross reacted with the ML-IAP epitope. This early evidence of a target for auto-reactive CTL in SS and SLE patients; it is to our knowledge previously unreported and underscores the important role of CD8 T cells in autoimmune disorders. Furthermore, the cross-reactivity against the auto-antigen SS56 and the tumor-antigen ML-IAP confirms the link between autoimmunity and anti-cancer cellular immune responses.

Efficient evaluation of humoral immune responses by the use of serum pools

BACKGROUND Collection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. METHODS We created serum pools from four different patient groups (systemic lupus erythematosus n=85, rheumatoid arthritis n=77, Sjögrens syndrome n=91, systemic sclerosis n=66) and one healthy control group (n=67). Each serum pool was analyzed using three well-known immunoassays: enzyme-linked immunosorbent assay (ELISA), line blot, and immunofluorescence microscopy (anti-nuclear antibody (ANA) screening). The presence of Epstein-Barr virus (EBV) EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies was used to validate serum pools as an efficient tool for further investigations by comparison to previous findings in this area. RESULTS The presence of EBV EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies in each pool was consistent within the obtained ELISA and line blot results, as increased titers of IgG against the four antigens were found in all patient serum pools and also in individual sera regarding gp350. These results correspond to previous findings on individual samples from patients with these diseases. The presence of ANAs was observed in all four patient serum pools and not in the HC pool by both line blots and immunofluorescence microscopy, which corresponds with the expectations and further corroborate the application of serum pools for screenings. CONCLUSION We developed and validated the use of serum pools that reliably and rapidly can confirm or deny hypotheses, which enables a more efficient research concentrating on the most evident factors.