T.R.D.J. Radstake

Formation of antibodies against infliximab and adalimumab strongly correlates with functional drug levels and clinical responses in rheumatoid arthritis.

Background: Tumour necrosis factor α (TNFα) neutralising antibody constructs are increasingly being used to treat rheumatoid arthritis (RA). Objective: To determine potential differences in clinical responses, soluble drug levels and antibody formation between patients with RA receiving infliximab and adalimumab. Methods: 69 patients with RA fulfilling the 1987 American College of Rheumatology criteria and about to start treatment with infliximab or adalimumab, were enrolled consecutively. All patients had active disease (28-joint count Disease Activity Score >3.2). Infliximab was given intravenously at 3 mg/kg at baseline and after 2, 6 and 14 weeks. Adalimumab was administered as 40 mg biweekly subcutaneously. Concomitant drug treatment was monitored and continued at constant dosage during the study. All serum samples were tested for infliximab/adalimumab levels and anti-infliximab/anti-adalimumab antibodies. Results: 35 patients received infliximab, 34 received adalimumab. At 6 months, 15 (43%), 6 (17%) and 14 (40%) of the infliximab-treated patients fulfilled the EULAR criteria for good, moderate and non-responders, respectively, whereas the corresponding figures for adalimumab-treated patients were 16 (47%), 8 (24%) and 10 (29%). Clinical responses correlated with the levels of S-infliximab/adalimumab and the formation of anti-infliximab/anti-adalimumab antibodies. Conclusion: The clinical response to two anti-TNFα biological agents closely follows the trough drug levels and the presence of antibodies directed against the drugs. Further studies that focus on the underlying pathways leading to antibody formation are warranted to predict immunogenicity of these expensive biological agents and treatment outcomes.

The STAT4 gene influences the genetic predisposition to systemic sclerosis phenotype

The aim of this study was to investigate the possible role of STAT4 gene in the genetic predisposition to systemic sclerosis (SSc) susceptibility or clinical phenotype. A total of 1317 SSc patients [896 with limited cutaneous SSc (lcSSc) and 421 with diffuse cutaneous SSc (dcSSc)] and 3113 healthy controls, from an initial case-control set of Spanish Caucasian ancestry and five independent cohorts of European ancestry (The Netherlands, Germany, Sweden, Italy and USA), were included in the study. The rs7574865 polymorphism was selected as STAT4 genetic marker. We observed that the rs7574865 T allele was significantly associated with susceptibility to lcSSc in the Spanish population [P = 1.9 x 10(-5) odds ratio (OR) 1.61 95% confidence intervals (CI) 1.29-1.99], but not with dcSSc (P = 0.41 OR 0.84 95% CI 0.59-1.21). Additionally, a dosage effect was observed showing individuals with rs7574865 TT genotype higher risk for lcSSc (OR 3.34, P = 1.02 x 10(-7) 95% CI 2.11-5.31). The association of the rs7574865 T allele with lcSSc was confirmed in all the replication cohorts with different effect sizes (OR ranging between 1.15 and 1.86), as well as the lack of association of STAT4 with dcSSc. A meta-analysis to test the overall effect of the rs7574865 polymorphism showed a strong risk effect of the T allele for lcSSc susceptibility (pooled OR 1.54 95% CI 1.36-1.74; P < 0.0001). Our data show a strong and reproducible association of the STAT4 gene with the genetic predisposition to lcSSc suggesting that this gene seems to be one of the genetic markers influencing SSc phenotype.

Anti-inflammatory therapy with tumour necrosis factor alpha inhibitors improves high-density lipoprotein cholesterol antioxidative capacity in rheumatoid arthritis patients

Objective: High-density lipoprotein (HDL) antiatherogenic functions seem to be diminished during inflammatory conditions such as rheumatoid arthritis (RA). The aim of this study was to investigate the effects of tumour necrosis factor (TNF) inhibition on the antioxidative capacity of HDL in RA. Methods: Plasma lipids and paraoxonase (PON-1) activity were investigated in 45 RA patients, before and during 6 months of anti-TNF therapy. In addition, HDL was isolated and tested for its ability to inhibit copper-induced oxidation of low-density lipoprotein in vitro. Results: Plasma HDL concentrations did not change considerably after 6 months of therapy. However, stable increases of PON-1 activities were observed throughout the same period (p<0.03). The increases were more obvious when related to HDL or apolipoprotein AI concentrations. HDL total antioxidative capacity significantly improved 6 months after the initiation of anti-TNF therapy (p = 0.015). The initial improvement of PON-1 activity paralleled a decrease in the inflammatory status, whereas specific TNF blockade was likely to be responsible for the long-term effects. Conclusions: Anti-TNF therapy with infliximab has beneficial effects on lipids through changes in HDL antioxidative capacity, which might be clinically relevant and contribute to the reported protective effect of anti-TNF on cardiovascular morbidity in RA. This emphasises the importance of HDL antiatherogenic capacity for cardiovascular risk in chronic inflammatory conditions.

Linkage proof for PTPN22, a rheumatoid arthritis susceptibility gene and a human autoimmunity gene

The tyrosine phosphatase PTPN22 allele 1858T has been associated with rheumatoid arthritis (RA) and other autoimmune diseases. RA is the most frequent of those multifactorial diseases. The RA association was usually restricted to serum rheumatoid factor positive disease (RF+). No interaction was shown with HLA-DRB1, the first RA gene. Many case-control studies replicated the RA association, showing an allele frequency increase of ≈5% on average and large variations of population allele frequencies (2.1–15.5%). In multifactorial diseases, the final proof for a new susceptibility allele is provided by departure from Mendels law (50% transmission from heterozygous parents). For PTPN22–1858T allele, convincing linkage proof was available only for type 1 diabetes. We aimed at providing this proof for RA. We analyzed 1,395 West European Caucasian individuals from 465 “trio” families. We replicated evidence for linkage, demonstrating departure from Mendels law in this subset of early RA onset patients. We estimated the overtransmission of the 1858T allele in RF+ families: T = 63%, P < 0.0007. The 1858T allele frequency increased from 11.0% in controls to 17.4% in RF+ RA for the French Caucasian population and the susceptibility genotype (1858T/T or T/C) from 20.2% to 31.6% [odds ratio (OR) = 1.8 (1.2–2.8)]. In conclusion, we provided the linkage proof for the PTPN22–1858T allele and RF+ RA. With diabetes and RA, PTPN22 is therefore a “linkage-proven” autoimmunity gene. PTPN22 accounting for ≈1% of the RA familial aggregation, many new genes could be expected that are as many leads to definitive therapy for autoimmune diseases.

BANK1 functional variants are associated with susceptibility to diffuse systemic sclerosis in Caucasians

Objective To investigate the possible association of the BANK1 gene with genetic susceptibility to systemic sclerosis (SSc) and its subphenotypes. Methods A large multicentre case–control association study including 2380 patients with SSc and 3270 healthy controls from six independent case–control sets of Caucasian ancestry (American, Spanish, Dutch, German, Swedish and Italian) was conducted. Three putative functional BANK1 polymorphisms (rs17266594 T/C, rs10516487 G/A, rs3733197 G/A) were selected as genetic markers and genotyped by Taqman 5´ allelic discrimination assay. Results A significant association of the rs10516487 G and rs17266594 T alleles with SSc susceptibility was observed (pooled OR=1.12, 95% CI 1.03 to 1.22; p=0.01 and pooled OR=1.14, 95% CI 1.05 to 1.25; p=0.003, respectively), whereas the rs3733197 genetic variant showed no statistically significant deviation. Stratification for cutaneous SSc phenotype showed that the BANK1 rs10516487 G, rs17266594 T and rs3733197 G alleles were strongly associated with susceptibility to diffuse SSc (dcSSc) (pooled OR=1.20, 95% CI 1.05 to 1.37, p=0.005; pooled OR=1.23, 95% CI 1.08 to 1.41, p=0.001; pooled OR=1.15, 95% CI 1.02 to 1.31, p=0.02, respectively). Similarly, stratification for specific SSc autoantibodies showed that the association of BANK1 rs10516487, rs17266594 and rs3733197 polymorphisms was restricted to the subgroup of patients carrying anti-topoisomerase I antibodies (pooled OR=1.20, 95% CI 1.02 to 1.41, p=0.03; pooled OR=1.24, 95% CI 1.05 to 1.46, p=0.01; pooled OR=1.26, 95% CI 1.07 to 1.47, p=0.004, respectively). Conclusion The results suggest that the BANK1 gene confers susceptibility to SSc in general, and specifically to the dcSSc and anti-topoisomerase I antibody subsets.

Type I interferons might form the link between Toll-like receptor (TLR) 3/7 and TLR4-mediated synovial inflammation in rheumatoid arthritis (RA)

Background: Rheumatoid arthritis (RA) has been associated with an increased risk of infections, but the underlying pathways have not yet been identified. Toll-like receptors (TLR) probably play a role in synovial inflammation and may also contribute to the understanding of the role of infections in RA. Objectives: To investigate if the synovial expression of TLR3 and TLR7 in RA correlates with that of inflammatory cytokines, and to assess whether this has functional consequences for local cytokine production and to study potential links between the TLR3/7 axis and TLR4 in RA synovium. Methods: Immunohistochemistry was used to study the expression of TLR3, TLR7, interferon α (IFNα), tumour necrosis factor α (TNFα) and interleukins IL1β, IL12, IL17 and IL18 in RA synovium obtained by arthroscopy from 34 patients with RA. Monocytes, monocyte-derived dendritic cells (MoDCs) and RA synovial fibroblasts were stimulated via TLR3 (poly-IC) and TLR7 (loxorubin), after which IL1β, IL6 and TNFα were measured by Luminex bead array technology. Following preincubation with IFNα, IL1β and IL18, TLR3 and TLR7 mRNA expression was assessed using real-time PCR. Cytokine production after preincubation with IFNα and subsequent TLR stimulation was measured. Results: Synovial TLR3/7 expression was co-expressed with IFNα, IL1β and IL18, but not with TNFα, IL12 and IL17. Stimulation of TLR3/TLR7 on monocytes, MoDCs or synovial fibroblasts led to secretion of type I IFN but no biologically active IL1β or IL18 could be detected. Type I IFNα increased TLR3/7 mRNA expression whereas IL1β and IL18 did not. In spite of the fact that the mRNA level of TLR4 remained unchanged, IFNα enhanced the response to TLR4 agonists, a phenomenon that was clearly more marked in patients with RA. Conclusion: Type I interferons are highly co-expressed with TLR3/TLR7 in RA synovium. They enhance TLR3/TLR7-mediated cytokine production and also TLR4-mediated responses.

Circulating leptin and adiponectin concentrations during tumor necrosis factor blockade in patients with active rheumatoid arthritis.

Objective. Adipocytokines, including leptin and adiponectin, may play an important role in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of longterm therapeutic tumor necrosis factor (TNF) blockade on adipocytokine concentrations in patients with RA. Methods. We studied 58 RA patients starting anti-TNF therapy and 58 healthy controls matched for age, sex, and body mass index (BMI). Fasting blood samples were drawn at baseline, 2 weeks, and 6 months after the start of anti-TNF therapy and serum levels of leptin and adiponectin were measured. Results. Patients with RA had increased adiponectin (p < 0.001) and similar leptin concentrations compared with the controls. Leptin concentrations were significantly higher in patients with high BMI (p < 0.001) and correlated positively with BMI at all timepoints (r > 0.75). In contrast, serum adiponectin tended to be higher in lean RA patients and did not correlate with BMI at any timepoint. There were no clear correlations between serum concentrations of adipocytokines and disease activity (Disease Activity Score 28). Short or longterm TNF blockade alone had no influence on circulating leptin and adiponectin concentrations. Patients treated with anti-TNF and concomitant corticosteroids on a stable basis showed a significant decrease in adiponectin levels after 6 months of therapy (p < 0.025). Conclusion. In patients with RA, chronic inflammation and its suppression during anti-TNF therapy have limited influence on plasma leptin concentrations, while significantly decreasing circulating adiponectin levels. Our findings question the suggested key role of inflammatory markers in regulating adipocytokine patterns in RA.

A rare polymorphism in the gene for Toll-like receptor 2 is associated with systemic sclerosis phenotype and increases the production of inflammatory mediators

OBJECTIVE To investigate whether polymorphisms in Toll-like receptor (TLR) genes, previously reported to be associated with immune-mediated diseases, are involved in systemic sclerosis (SSc). METHODS We genotyped 14 polymorphisms in the genes for TLRs 2, 4, 7, 8, and 9 in a discovery cohort comprising 452 SSc patients and 537 controls and a replication cohort consisting of 1,170 SSc patients and 925 controls. In addition, we analyzed 15-year followup data on 964 patients to assess the potential association of TLR variants with the development of disease complications. We analyzed the functional impact of the associated polymorphism on monocyte-derived dendritic cells. RESULTS In the discovery cohort, we observed that a rare functional polymorphism in TLR2 (Pro631His) was associated with antitopoisomerase (antitopo) positivity (odds ratio 2.24 [95% confidence interval 1.24-4.04], P=0.003). This observation was validated in the replication cohort (odds ratio 2.73 [95% confidence interval 1.85-4.04], P=0.0001). In addition, in the replication cohort the TLR2 variant was associated with the diffuse subtype of the disease (P=0.02) and with the development of pulmonary arterial hypertension (PAH) (Cox proportional hazards ratio 5.61 [95% confidence interval 1.53-20.58], P=0.003 by log rank test). Functional analysis revealed that monocyte-derived dendritic cells carrying the Pro63His variant produced increased levels of inflammatory mediators (tumor necrosis factor α and interleukin-6) upon TLR-2-mediated stimulation (both P<0.0001). CONCLUSION Among patients with SSc, the rare TLR2 Pro631His variant is robustly associated with antitopoisomerase positivity, the diffuse form of the disease, and the development of PAH. In addition, this variant influences TLR-2-mediated cell responses. Further research is needed to elucidate the precise role of TLR-2 in the pathogenesis of SSc.

Analysis of the influence of PTPN22 gene polymorphisms in systemic sclerosis

Objective Two functional single nucleotide polymorphisms (SNP) in the PTPN22 gene (rs24746601 and rs33996649) have been associated with autoimmunity. The aim of this study was to investigate the role of the R263Q SNP for the first time and to re-evaluate the role of the R620W SNP in the genetic predisposition to systemic sclerosis (SSc) susceptibility and clinical phenotypes. Methods 3422 SSc patients (2020 with limited cutaneous SSc and 1208 with diffuse cutaneous SSc) and 3638 healthy controls of Caucasian ancestry from an initial case--control set of Spain and seven additional independent replication cohorts were included in our study. Both rs33996649 and rs2476601 PTPN22 polymorphisms were genotyped by TaqMan allelic discrimination assay. A meta-analysis was performed to test the overall effect of these PTPN22 polymorphisms in SSc. Results The meta-analysis revealed evidence of association of the rs2476601 T allele with SSc susceptibility (pFDRcorrected=0.03 pooled, OR 1.15, 95% CI 1.03 to 1.28). In addition, the rs2476601 T allele was significantly associated with anticentromere-positive status (pFDRcorrected=0.02 pooled, OR 1.22, 95% CI 1.05 to 1.42). Although the rs33996649 A allele was significantly associated with SSc in the Spanish population (pFDRcorrected=0.04, OR 0.58, 95% CI 0.36 to 0.92), this association was not confirmed in the meta-analysis (p=0.36 pooled, OR 0.89, 95% CI 0.72 to 1.1). Conclusion The study suggests that the PTPN22 R620W polymorphism influences SSc genetic susceptibility but the novel R263Q genetic variant does not. These data strengthen evidence that the R620W mutation is a common risk factor in autoimmune diseases.

The TRAF1-C5 region on chromosome 9q33 is associated with multiple autoimmune diseases.

Objectives The TRAF1-C5 locus has recently been identified as a genetic risk factor for rheumatoid arthritis (RA). Since genetic risk factors tend to overlap with several autoimmune diseases, a study was undertaken to investigate whether this region is associated with type 1 diabetes (TID), celiac disease (CD), systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). Methods The most consistently associated SNP, rs10818488, was genotyped in a total of 735 patients with T1D, 1049 with CD, 367 with SSc, 746 with SLE and 3494 ethnically- and geographically-matched healthy individuals. The replication sample set consisted of 99 patients with T1D, 272 with SLE and 482 healthy individuals from Crete. Results A significant association was detected between the rs10818488 A allele and T1D (OR 1.14, p=0.027) and SLE (OR 1.16, p=0.016), which was replicated in 99 patients with T1D, 272 with SLE and 482 controls from Crete (OR 1.64, p=0.002; OR 1.43, p=0.002, respectively). Joint analysis of all patients with T1D (N=961) and all patients with SLE (N=1018) compared with 3976 healthy individuals yielded an allelic common OR of 1.19 (p=0.002) and 1.22 (p=2.6×10−4), respectively. However, combining our dataset with the T1D sample set from the WTCCC resulted in a non-significant association (OR 1.06, p=0.087). In contrast, previously unpublished results from the SLEGEN study showed a significant association of the same allele (OR 1.19, p=0.0038) with an overall effect of 1.22 (p=1.02×10−6) in a total of 1577 patients with SLE and 4215 healthy individuals. Conclusion A significant association was found for the TRAF1-C5 locus in SLE, implying that this region lies in a pathway relevant to multiple autoimmune diseases.