cDNA phage display for the discovery of theranostic autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the world's most common autoimmune disease mainly characterized by a chronic inflammation of multiple synovial joints. Rheumatologists now have a whole range of treatment options including glucocorticoids (GCs), classical synthetic and biological disease-modifying antirheumatic drugs (cs- and bDMARDS), resulting in a tremendous improvement in treatment outcomes for RA patients over the last two decades. Despite this progress, the choice of treatment regimen to achieve stable remission at the individual patient level still largely depends on trial and error. In this review, the need for novel theranostic markers that can predict a patient's response to methotrexate, the standard first-line csDMARD treatment, is discussed. Like in many autoimmune diseases, the majority of RA patients form a whole range of autoantibodies. We aim to find novel theranostic autoantibody markers using serological antigen selection, a high-throughput technique that uses cDNA phage display to identify novel antigen targets. We have constructed a barcoded cDNA phage display library from the synovial tissue of three RA patients by fusing cDNA products to the filamentous phage minor coat protein VI. This library contains a large proportion of full-length genes and gene fragments that are cloned in frame with the phage gene VI. By screening this library for antibody reactivity in serum samples of patients from the CareRA trial, which compared different intensive treatment strategies based on csDMARDs and a step-down GC schedule, our cDNA phage display library has great potential for the discovery of novel theranostic autoantibody biomarkers.

Detection of novel diagnostic antibodies in ankylosing spondylitis: An overview.

Ankylosing spondylitis (AS) is a debilitating, chronic, rheumatic disease characterized by inflammation and new bone formation resulting in fusion of the spine and sacroiliac joints. Since early treatment is impeded by a delayed diagnosis, it is highly important to find new biomarkers that improve early diagnosis and may also contribute to a better assessment of disease activity, prognosis and therapy response in AS. Because of the absence of rheumatoid factor, AS was long assumed to have a seronegative character and antibodies are thus not considered a hallmark of the disease. However, emerging evidence suggests plasma cells and autoantibodies to be involved in the disease course. In this review, the role of B cells and antibodies in AS is discussed. Furthermore, an overview is provided of antibodies identified in AS up till now, and their diagnostic potential. Many of these antibody responses were based on small study populations and further validation is lacking. Moreover, most were identified by a hypothesis-driven approach and thus limited to antibodies against targets that are already known to be involved in AS pathogenesis. Hence, we propose an unbiased approach to identify novel diagnostic antibodies. The already successfully applied techniques cDNA phage display and serological antigen selection will be used to identify antibodies against both known and new antigen targets in AS plasma. These newly identified antibodies will enhance early diagnosis of AS and provide more insight into the underlying disease pathology, resulting in a more effective treatment strategy and eventually an improved disease outcome.

The isotype repertoire of antibodies against novel UH-RA peptides in rheumatoid arthritis.

BACKGROUND: Recently, autoantibodies against novel UH-RA peptides (UH-RA.1 and UH-RA.21) were identified as candidate biomarkers for patients with rheumatoid arthritis (RA) who are seronegative for the current diagnostic markers rheumatoid factor and anticitrullinated protein antibodies. Previously, screening for anti-UH-RA autoantibodies was based on measuring the immunoglobulin (Ig) G response. We aimed to investigate whether measurement of other isotypes could improve the performance of diagnostic testing. In addition, assigning the isotype profile might provide valuable information on effector functions of the antibodies. METHODS: The isotype profile of antibodies against UH-RA.1 and UH-RA.21 was studied. The IgG, IgM, and IgA classes, together with the 4 different IgG subclasses, were determined in 285 patients with RA, 88 rheumatic control subjects, and 90 healthy control subjects. RESULTS: Anti-UH-RA.1 antibodies were primarily of the IgM isotype and twice as prevalent as IgG (IgG3-dominated) and IgA. RA sensitivity when testing for anti-UH-RA.1 IgM was shown to be higher than when testing for the IgG isotype: 18 % versus 9 % sensitivity when RA specificity was set to 90 %. Within antibodies against UH-RA.21, IgG and IgA were more common than IgM. Different anti-UH-RA.21 IgG subclasses were found, with the highest prevalence found for IgG2. Combined testing for IgG and IgA slightly increased RA sensitivity of UH-RA.21-specific antibody testing to 27 % compared with solely testing for IgG (23 %). Notably, a higher number of anti-UH-RA.21 antibody isotypes was related to increased levels of erythrocyte sedimentation rate. Finally, for both antibody responses, the full antibody isotype use was demonstrated in early and seronegative disease. CONCLUSIONS: The isotype distribution of anti-UH-RA.1 and anti-UH-RA.21 antibodies was successfully outlined, and, for antibodies against UH-RA.1, we found that isotype-specific testing might have implications for diagnostic testing. The exact mechanisms by which the different antibody isotypes act still have to be unraveled.

Autoantibodies to two novel peptides in seronegative and early rheumatoid arthritis.

OBJECTIVES: Despite recent progress in biomarker discovery for RA diagnostics, still over one-third of RA patients-and even more in early disease-present without RF or ACPA. The aim of this study was to confirm the presence of previously identified autoantibodies to novel Hasselt University (UH) peptides in early and seronegative RA. METHODS: Screening for antibodies against novel UH peptides UH-RA.1, UH-RA.9, UH-RA.14 and UH-RA.21, was performed in two large independent cohorts. Peptide ELISAs were developed to screen for the presence of antibodies to UH-RA peptides. First, 292 RA patients (including 39 early patients), 90 rheumatic and 97 healthy controls from UH were studied. Antibody reactivity to two peptides (UH-RA.1 and UH-RA.21) was also evaluated in 600 RA patients, 309 patients with undifferentiated arthritis and 157 rheumatic controls from the Leiden Early Arthritis Clinic cohort. RESULTS: In both cohorts, 38% of RA patients were seronegative for RF and ACPA. Testing for autoantibodies to UH-RA.1 and UH-RA.21 reduced the serological gap from 38% to 29% in the UH cohort (P = 0.03) and from 38% to 32% in the Leiden Early Arthritis Clinic cohort (P = 0.01). Furthermore, 19-33% of early RA patients carried antibodies to these peptides. Specificities in rheumatic controls ranged from 82 to 96%. Whereas antibodies against UH-RA.1 were related to remission, anti-UH-RA.21 antibodies were associated with inflammation, joint erosion and higher tender and swollen joint counts. CONCLUSION: This study validates the presence of antibody reactivity to novel UH-RA peptides in seronegative and early RA. This might reinforce current diagnostics and improve early diagnosis and intervention in RA.

The prevalence of vertebral fractures in spondyloarthritis: relation to disease characteristics, bone mineral density, syndesmophytes and history of back pain and trauma.

INTRODUCTION: An increased risk of vertebral fracture (VF) is one of the extra-articular manifestations of spondyloarthropathy (SpA). The prevalence of moderate to severe VFs visualized by radiography (Rx) in patients with SpA in daily practice is unknown until imaging of the full spine is available, as most VFs do not present with clinical signs and symptoms of an acute fracture. METHODS: We evaluated the prevalence of VFs (>25% loss in height) on available Rx and dual-energy X-ray absorptiometry (DXA) images in 390 consecutive patients with SpA in daily practice. We assessed their association with disease characteristics, bone mineral density, the modified Stoke Ankylosing Spondylitis Spinal Score, and history of trauma. RESULTS: Forty-six patients (11.8%) had Rx VF (56.4% men, 93.5% in the thoracic spine), and 44.5% had multiple VFs. Compared with patients without VF, patients with VF were older (52.2 vs. 47.3 years, p < 0.01; range 25-84 years), had lower femoral neck T-scores (-1.1 vs. -0.7; p < 0.05), and had a marginally higher modified Stoke Ankylosing Spondylitis Spinal Score (11.7 vs. 7.0; p = 0.06). Among patients with VFs, 15.2% had a history of trauma with acute back pain (p < 0.001 vs. no VF). The reliability of DXA for diagnosing radiographic VFs was high (κ 0.90). CONCLUSIONS: Moderate to severe VFs are found in more than 10% of patients with SpA before the age of 40 years in 5% of women and 9% in men. Most VFs are located in the thoracic region, are related to low femoral neck bone mineral density and to stiffening of the spine, and are only rarely related to trauma history. DXA is a useful alternative for diagnosing VFs.

The association between urinary kidney injury molecule 1 and urinary cadmium in elderly during long-term, low-dose cadmium exposure: a pilot study.

BACKGROUND: Urinary kidney injury molecule 1 is a recently discovered early biomarker for renal damage that has been proven to be correlated to urinary cadmium in rats. However, so far the association between urinary cadmium and kidney injury molecule 1 in humans after long-term, low-dose cadmium exposure has not been studied. METHODS: We collected urine and blood samples from 153 non-smoking men and women aged 60+, living in an area with moderate cadmium pollution from a non-ferrous metal plant for a significant period. Urinary cadmium and urinary kidney injury molecule 1 as well as other renal biomarkers (alpha1-microglobulin, beta2-microglobulin, blood urea nitrogen, urinary proteins and microalbumin) were assessed. RESULTS: Both before (r = 0.20; p = 0.01) and after (partial r = 0.32; p < 0.0001) adjustment for creatinine, age, sex, past smoking, socio-economic status and body mass index, urinary kidney injury molecule 1 correlated with urinary cadmium concentrations. No significant association was found between the other studied renal biomarkers and urinary cadmium. CONCLUSIONS: We showed that urinary kidney injury molecule 1 levels are positively correlated with urinary cadmium concentration in an elderly population after long-term, low-dose exposure to cadmium, while other classical markers do not show an association. Therefore, urinary kidney injury molecule 1 might be considered as a biomarker for early-stage metal-induced kidney injury by cadmium.

Effect of pH on the stability of kidney injury molecule 1 (KIM-1) and on the accuracy of its measurement in human urine.

BACKGROUND: Urinary KIM-1 is a novel biomarker for tubular kidney damage, however little is known about its stability. The goal of this study is to examine the effect of urinary pH on the stability of KIM-1. METHODS: Urine samples were collected from 45 volunteers. Samples were aliquoted, adapted to different pH values (range 4 to 9) and stored at -80°C. After thawing, each aliquot was divided into two, of which one was used to measure KIM-1 (human tim-1/kim-1/Havcr Elisa kit; R&D systems) at the same pH at which it was stored, while the other was readapted to pH 7 before measurement. RESULTS: KIM-1 values of aliquots of the same sample are stable when stored at pH 6, 7 and 8 whereas at lower and higher storage pH, KIM-1 levels decrease significantly. When samples are readjusted to a neutral pH just before KIM-1 measurement, there are no longer significant differences between KIM-1 in aliquots stored at different pH values. CONCLUSIONS: No effect of urinary pH on the stability of KIM-1 was seen. However, the only commercially available human tim-1/kim-1/Havcr Elisa kit of RD systems is pH dependent and we therefore suggest samples should be adjusted to neutral pH before measurement.