Anna Polgár

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases.

Introduction Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. Methods In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. Results EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3+ and CD8+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction). Conclusions Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

Cost-of-illness of patients with systemic sclerosis in a tertiary care centre

OBJECTIVE The aim of our study was to assess the costs of SSc and to analyse cost drivers. METHODS A cross-sectional survey of consecutive patients with SSc was performed in a rheumatology centre in Hungary. Clinical characteristics, the European Scleroderma Study Group activity index, disease severity scale (DSS), scleroderma HAQ (S-HAQ) and health care utilization were recorded. Cost calculation was performed and correlation with clinical variables was analysed. Results were compared with RA and PsA. RESULTS Eighty patients were involved: 72 (90%) women, mean age (s.d.) 57.4 (9.6) years and disease duration of 6.2 (6.6) years and 25% of the cases had dcSSc. Mean total cost was 9619 (s.d. 6444) euros/patient/year with rate of indirect cost being 56%. Disability-related productivity loss (55.2%) and hospitalization (28.3%) were the highest among the cost items. Patients with dcSSc had significantly higher direct costs (P = 0.005) compared with the lcSSc subset. Disease activity showed significant correlation with total costs, DSS and S-HAQ with direct costs. SSc-related costs were higher than in matched RA and PsA cases. CONCLUSIONS The cost-of-illness of SSc is high with a dominance of productivity loss related costs. Moreover, the disease activity is an important cost driver.

Natural autoantibodies reactive with glycosaminoglycans in rheumatoid arthritis.

IntroductionAlthough natural autoantibodies make up the majority of circulating immunoglobulins and are also present in high numbers in therapeutically used intravenous immunoglobulin preparations, they have received little attention and their precise role remains largely unknown. An increasing awareness of the importance of posttranslational autoantigen modifications and glycobiology led us to explore carbohydrate-reactive natural autoantibodies in patients with rheumatoid arthritis. This study examined systematic antibodies reactive to glycosaminoglycans (GAGs), the carbohydrate components of proteoglycans that are released in large amounts from degrading cartilage.MethodsTo measure antibodies reactive to six different types of GAGs, a specialised ELISA was used in which the carbohydrates were covalently linked to the plastic surface through a 2 nm spacer. Sera from rheumatoid arthritis patients (n = 66), umbilical cord serum samples (n = 11) and adult controls (n = 54) were studied. In order to explore cross-reactivity with microbial antigens, bacterial peptidoglycans and fungal polysaccharides were used. Sera and synovial fluid samples were also tested using a GlycoChip carbohydrate array to characterise individual carbohydrate recognition patterns. We followed a multistep statistical screening strategy for screening GAG-reactive antibodies as predictive disease markers.ResultsWhile anti-GAG antibodies were absent in the umbilical cord sera, they were readily detectable in adult controls and were significantly elevated in patients with rheumatoid arthritis (p < 0.001). Anti-GAG antibodies showed significant cross-reactivity among different types of GAGs. They also reacted with bacterial peptidoglycans and fungal polysaccharides. Interestingly, anti-chondroitin sulphate C IgM antibody levels showed inverse correlation both with the Disease Activity Score (DAS) 28 scores and C-reactive protein (CRP) levels in rheumatoid arthritis.ConclusionThe highly abundant and cross-reactive, GAG-specific natural autoantibodies in serum may serve as novel disease-state markers in patients with rheumatoid arthritis.

Comparison of the Psoriatic Arthritis Quality of Life (PsAQoL) questionnaire, the functional status (HAQ) and utility (EQ-5D) measures in psoriatic arthritis: results from a cross-sectional survey

Objectives: To compare the Psoriatic Arthritis Quality of Life (PsAQoL) instrument, the Health Assessment Questionnaire (HAQ) as a measure of functional status, and the generic health status (utility) measure the EuroQoL (EQ-5D) in terms of ability to assess disease severity in psoriatic arthritis (PsA). Methods: The differences between known groups and correlations of the PsAQoL, the HAQ and the EQ-5D with clinical measures were analysed in a sample of 183 PsA patients. Results: Different severities of PsA determined by known groups were distinguished well by all three questionnaires; more severe disease was associated with significantly worse values of the instruments. The correlations revealed a strong relationship between each of the measures, and with the patients’ pain on the visual analogue scale (VAS), the patient global VAS, and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and a weak relationship with the disease duration and the Psoriasis Area Severity Index (PASI). The PsAQoL also correlated strongly with the 28-joint Disease Activity Score (DAS28). Conclusions: The PsAQoL, the HAQ, and the EQ-5D are able to distinguish well across levels of PsA severity.

Complement receptor type 1 (CR1, CD35) is a potent inhibitor of B-cell functions in rheumatoid arthritis patients

The involvement of B cells, complement activation and subsequent immune complex deposition has all been implicated in the pathogenesis of rheumatoid arthritis (RA). Although the reduced expression of complement receptor 1 (CR1, CD35) and 2 (CR2, CD21) on the B cells of RA patients has been known for a long time, their exact role in B-cell tolerance and autoimmunity is not yet fully understood. To get a deeper insight into the possible mechanisms, we studied the expression and function of CR1 and CR2 on various subsets of B cells of healthy donors and RA patients at various stages of the disease by FACS analysis, (3)H-thymidine incorporation and ELISA. We found that CD19(+)CD27(-) naive B cells up-regulate the expression of the inhibitory CR1 during differentiation to CD19(+)CD27(+) memory B cells both in healthy donors and in RA patients, whereas the expression of the activatory CR2 is down-regulated. This clearly demonstrates that the expression of these two antagonistic complement receptors is regulated differentially during the development of human B cells, a phenomenon which may influence the maintenance of peripheral B-cell tolerance. Our functional studies show that after clustering CR1 both by its natural ligand and To5 mAb, the inhibitory function of CD35 is maintained in RA patients, despite its significantly reduced expression compared with healthy individuals. Besides blocking B-cell receptor-induced proliferation, CR1 inhibits the differentiation of B cells to plasmablasts and their immunoglobulin production. Since the reduced expression of CR1 in RA patients does not affect its inhibitory function, this receptor might serve as a new target for therapeutical interventions.

Devic's syndrome and SLE. Challenges in diagnosis and therapeutic possibilities based on two overlapping cases.

Neuromyelitis optica (NMO, Devics disease), an uncommon demyelinating neuro-immunological disease, can be associated with autoimmune diseases. In SLE associated forms anti-aquaporin-4 antibody positivity can help differentiating between SLE nerve system manifestation and NMO. In the literature rituximab, or immunoablative dose cyclophosphamide (CYC) was effective for the therapy resistant forms. Authors present 2 SLE overlapping NMO cases, one of them with SLE associated interstitial lung disease (ILD). In both cases neurological manifestations anticipated other SLE symptoms. Patients previously were treated with high dose corticosteroid therapy, plasmapheresis, and one of them with azathioprine, and the other one with oral CYC (which could not prevent flares). 0.5 g/m² body-surface monthly parenteral inductive CYC therapy was administered, in one patient followed by quarterly maintenance therapy. This patient completed her 18 month maintenance treatment and has been in neurological remission, but required steroid pulse and plasmapheresis for lung symptoms. The second patient had urogenital infection after the induction phase, followed by an exacerbation, requiring plasmapheresis and high dose parenteral corticosteroid treatment. After it he refused CYC therapy and has been taking azathioprine. He has no new symptoms, only residual ones. In our two patients conventional dose CYC therapy proved to be effective for NMO/SLE overlap, required only transient supportive therapy.

Generation of 'truncated' interleukin-6 receptor (IL-6R) mRNA by alternative splicing; a possible source of soluble IL-6R.

Receptors for interleukin-6 (IL-6) occur in body fluids in soluble form, as well. This is an approx. 50 kDa protein with the ability to bind IL-6. The soluble IL-6 receptor (sIL-6R)/IL-6 complex can attach to membrane anchored gp130, a molecule associated with the signal transduction induced by IL-6 and by other related cytokines. Earlier we described the appearance of sIL-6R in various body fluids of autoimmune patients. In this study using reverse transcriptase polymerase chain reaction (RT-PCR) we isolated and characterised a truncated form of amplified cDNA reverse-transcribed from IL-6 receptor mRNA both from human hepatoma cell line HepG2 and mononuclear cells from inflammatory bowel disease (IBD) patients. Using digestion by Pvu II restriction endonuclease and direct nucleotide sequencing we conclude that alternative splicing is likely involved in generation of sIL-6R. Our further experiments suggest that IL-6 and recombinant sIL-6R themselves do not influence the alternative splicing of IL-6 receptor gene.

Soluble interleukin-6 receptor enhanced by oncostatin M induces major changes in gene expression profile of human hepatoma cells

Interleukin-6 (IL-6) binds to a receptor complex consisting of an 80 kDa binding unit (IL-6R) and gp130 responsible for signal transduction. Due to alternative splicing and/or proteolytic digestion IL-6R occurs in soluble form (sIL-6R), as well. Soluble IL-6R is able to bind to gp130 expressing on nucleated cells, thus sIL-6R makes most cells responsive to IL-6. In this study we found that oncostatin M (OSM), an other gp130 dependent cytokine with proliferation inhibitory potential, increases the expression of both membrane-bound IL-6R and sIL-6R generated by alternative splicing in hepatic and mammary carcinoma cell lines. Furthermore, we studied the functional relevance of the presence and binding of soluble IL-6R to HepG2 cells. Using a cDNA expression array, mRNA levels of about 580 human genes were tested by differential display analysis. Our findings suggest, that elevation of surface density of IL-6R by attachment of sIL-6R induces major modulation in gene expression profile of the hepatoma cells. Soluble IL-6R alone has minor effect, it rather decreases expression of some genes, while incubation with IL-6 and sIL-6R together induces major changes in the mRNA pattern of HepG2 cells. These data strongly suggest that presence and binding of soluble cytokine receptors are important elements of inter-cytokine cross talk and affects actual gene expression profile of responding cells.

Impact of disease progression on health status, quality of life and costs in rheumatoid arthritis in Hungary

UNLABELLED Rheumatoid arthritis (RA) is a chronic, progressive polyarthritis leading to substantial disability. Standardised data on consequences of disease progression are needed for clinical assessments and also for cost-effectiveness models. AIM To analyse the impact of disease progression on health status, disease specific quality of life and costs in Hungary. METHODS A cross-sectional survey was performed between April and August, 2004, involving consecutive RA patients of 6 hospital based rheumatology outpatient centres. Self-completed questionnaires were used to assess functional (HAQ) and health status (EQ-5D), quality of life (RAQoL). Disease activity (DAS) and costs were also surveyed, statistical analysis was performed. RESULTS 255 patients were involved [mean age 55.5 +/- 12.3 years; disease duration 9.0 +/- 9.3 years; HAQ 1.38 +/- 0.76; EQ-5D 0.46 +/- 0.33; RAQoL 16.2 +/- 8.1; DAS 5.09 +/- 1.42; costs 1,043,163 (+/- 844,750) HUF/patient/year, conversion 1 Euro = 250 HUF]. Correlation was significant between the parameters ( p < 0.01): EQ-5D index = 1.014 - 0.25 x HAQ-0.041 x DAS; HAQ = 0.314 + 0.065 x RAQoL. Analysis by disease severity levels (HAQ groups 0.5 difference) revealed that health status worsens (mean EQ-5D: 0.784; 0.576; 0.504; 0.367; 0.211; 0.022) and costs increase (mean 628,280; 888,187; 953,759; 1,291,218; 1,346,112; 1,371,674 HUF/patient/year) with disease progression. Minimally important worsening of functional ability (0.25 HAQ increase) corresponds to -0.0705 EQ-5D and +1.884 RAQoL change. Lower health status difference (EQ-5D -0.05725) was calculated in patients with lower disease activity (DAS < 5.1). CONCLUSIONS Correlation between disease progression, health status, quality of life and costs does not differ significantly from international results. The amount of costs is much lower in all disease severity levels than in developed European countries. Our study serves baseline data for health economic analysis in RA in Hungary.