Autoantibodies against complement factor B in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder affecting the joints. Many patients carry anti-citrullinated protein autoantibodies (ACPA). Overactivation of the complement system seems to be part of the pathogenesis of RA, and autoantibodies against the pathway initiators C1q and MBL, and the regulator of the complement alternative pathway, factor H (FH), were previously reported. Our aim was to analyze the presence and role of autoantibodies against complement proteins in a Hungarian RA cohort. To this end, serum samples of 97 ACPA-positive RA patients and 117 healthy controls were analyzed for autoantibodies against FH, factor B (FB), C3b, C3-convertase (C3bBbP), C1q, MBL and factor I. In this cohort, we did not detect any patient with FH autoantibodies but detected C1q autoantibodies in four patients, MBL autoantibodies in two patients and FB autoantibodies in five patients. Since the latter autoantibodies were previously reported in patients with kidney diseases but not in RA, we set out to further characterize such FB autoantibodies. The isotypes of the analyzed autoantibodies were IgG2, IgG3, IgGκ, IgGλ and their binding site was localized in the Bb part of FB. We detected in vivo formed FB-autoanti-FB complexes by Western blot. The effect of the autoantibodies on the formation, activity and FH-mediated decay of the C3 convertase in solid phase convertase assays was determined. In order to investigate the effect of the autoantibodies on complement functions, hemolysis assays and fluid phase complement activation assays were performed. The autoantibodies partially inhibited the complement-mediated hemolysis of rabbit red blood cells, inhibited the activity of the solid phase C3-convertase and C3 and C5b-9 deposition on complement activating surfaces. In summary, in ACPA-positive RA patients we identified FB autoantibodies. The characterized FB autoantibodies did not enhance complement activation, rather, they had inhibitory effect on complement. These results support the involvement of the complement system in the pathomechanism of RA and raise the possibility that protective autoantibodies may be generated in some patients against the alternative pathway C3 convertase. However, further analyses are needed to assess the exact role of such autoantibodies.

[COVID-19 infections and effectiveness of the vaccination among healthcare workers]. / A COVID­19-fertozés és a védooltások hatásosságának vizsgálata egészségügyi dolgozókon.

INTRODUCTION: New variants of the SARS-CoV-2 coronavirus are constantly appearing, causing the COVID-19 pandemic. From November 2021, most infections were caused by the Omicron coronavirus variant. OBJECTIVE: The aim of this prospective observational cohort study was to estimate the incidence of COVID-19 infections in the high-risk healthcare workers after two BNT162b2 mRNA Pfizer-BioNTech vaccines and the subsequent booster vaccine, as well as the effectiveness, the safety and the humoral immune response of the vaccines. METHOD: We started the two Pfizer-BioNTech ((BNT162b29) vaccinations of healthcare workers of the Polyclinic of the Hospitaller Brothers of St. John between January 07 and March 08, 2021. The choice of the type and timing of the third booster vaccination was voluntary. The workers were followed up between January 07, 2021 and June 29, 2022. The infection rate, adverse events of the vaccination, risk factors to infection and the kinetics of anti-spike (S) antibody and anti-nucleocapsid (N) antibody serum level were evaluated. RESULTS: The data of 294 healthcare workers - 96 medical doctors, 127 nurses and 71 workers in hospital - who had at least three antibody level measurements were analyzed. The third booster vaccine was given to 280 workers, the distribution of the vaccines was the following: Pfizer-BioNTech (BNT162b29) vaccine (n = 210), Moderna COVID-19 (mRNA-1273) vaccine (n = 37), Sinopharm COVID-19 vaccine (n = 21), Janssen COVID-19 (n = 10), AstraZeneca (ChAdOx1 nCoV-19) vaccine (n = 2). Infection occurred in 121 cases (41%) during the observation period. The course of the COVID-19 infections was mostly mild (97%) and recovered within a week. During the observational period, 2 workers died: a 56-year-old woman died after two vaccinations for reason unrelated to COVID-19 infection, and a 58-year-old man died after the booster vaccination, following COVID-19 infection. The incidence of infection did not correlate with age, sex, comorbidities, smoking, occupation and BMI. The median titre of anti-S antibody serum level increased one month after the second vaccination of the basic immunization (1173.0 U/ml) and decreased slowly until the 8th month (678.5-625.8-538.0 U/ml) after the basic vaccination. One month after the booster vaccination, the median titre of anti-S antibody serum level increased significantly (16 535 U/ml), and showed a decreasing trend in the 3rd month after the booster vaccination (9697.7 U/ml). An exceptionally high S antibody serum level increasing after the basic (>10 000 U/mL) and booster (>60 000 U/m) vaccination showed a correlation with prior COVID-19 infection. The median cut-off index (COI) of anti-N antibody was not affected by vaccination, the increasing of the titre is related to the infection. CONCLUSION: The booster vaccination had less effect on the infection caused by Omicron variant, but the course of the infection was milder. Compared to the basic immunisation, the booster vaccination caused a significant increase in the S antibody level. An exceptionally high S antibody level correlated with prior COVID-19 infection. Orv Hetil. 2023; 164(5): 163-171.

Efficacy and Safety of ABBV-3373, a Novel Anti-Tumor Necrosis Factor Glucocorticoid Receptor Modulator Antibody-Drug Conjugate, in Adults with Moderate-to-Severe Rheumatoid Arthritis Despite Methotrexate Therapy: A Randomized, Double-Blind, Active-Controlled Proof-of-Concept Phase IIa Trial.

OBJECTIVE: To assess the efficacy and safety of ABBV-3373, a novel antibody-drug conjugate (ADC) composed of the anti-tumor necrosis factor (anti-TNF) monoclonal antibody adalimumab linked to a glucocorticoid receptor modulator (GRM), compared to adalimumab, in patients with rheumatoid arthritis (RA). METHODS: In this randomized, double-blind, active-controlled, proof-of-concept trial (ClinicalTrials.gov identifier: NCT03823391), adults with moderate-to-severe RA receiving background methotrexate were administered intravenously (IV) ABBV-3373 100 mg every other week for 12 weeks, followed by placebo for 12 weeks, or subcutaneous adalimumab 80 mg every other week for 24 weeks. The primary end point was change from baseline in the Disease Activity Score in 28 joints using C-reactive protein (DAS28-CRP) at week 12, with 2 prespecified primary comparisons of ABBV-3373 versus historical adalimumab (80 mg every other week or equivalent dose) and versus combined in-trial/historical adalimumab. Secondary end points included change from baseline in the Clinical Disease Activity Index, Simplified Disease Activity Index, and DAS28 using erythrocyte sedimentation rate, as well as the proportion of patients achieving a DAS28-CRP of ≤3.2 and the American College of Rheumatology 50% improvement criteria. RESULTS: Forty-eight patients were randomized to receive either ABBV-3373 (n = 31) or adalimumab (n = 17). At week 12, ABBV-3373 demonstrated a reduction in DAS28-CRP compared to historical adalimumab (-2.65 versus -2.13; P = 0.022) and compared to combined in-trial/historical adalimumab (-2.65 versus -2.29; probability 89.9%), with numerically greater improvement than in-trial adalimumab (-2.51). For secondary end points, greater efficacy was observed with ABBV-3373 compared to historical adalimumab; ABBV-3373 was predicted with 79.3-99.5% probability to be more effective than adalimumab based on combined in-trial/historical adalimumab data. Of the ABBV-3373-treated patients who achieved DAS28-CRP ≤3.2 at week 12, 70.6% maintained this response at week 24 despite switching to placebo. Four serious adverse events (SAEs) were reported with ABBV-3373 (noncardiac chest pain, pneumonia, upper respiratory tract infection, and anaphylactic shock) and 2 SAEs with adalimumab (breast abscess and bronchitis). After increasing the duration of IV ABBV-3373 administration from 3 minutes to 15-30 minutes, no similar events of anaphylactic shock were reported. CONCLUSION: Data from this proof-of-concept trial support the continued development of a TNF-GRM ADC for the treatment of RA, with the potential to achieve superior outcomes compared to currently available therapies.

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

Immunogenicity and safety of the BNT162b2 mRNA COVID-19 vaccine in healthcare workers / A BNT162b2 mRNS-Pfizer-BioNTech-védooltás hatásosságának és immunogenitásának monitorozása egészségügyben dolgozókon.

Összefoglaló. Bevezetés: A SARS-CoV-2 koronavírus okozta COVID-19 általános egészségügyi és gazdasági krízist idézett elo. Célkituzés: A megfigyeléses vizsgálat célja a BNT162b2 mRNS-Pfizer-BioNTech-vakcina hatásosságának, biztonságosságának és immunogenitásának igazolása a Budai Irgalmasrendi Kórház dolgozóin. Módszer: A vakcina adása után elemeztük a COVID-19-fertozés elofordulását, az oltások utáni reakciókat, valamint a "spike" (S-) protein és a nukleokapszid (N)-protein elleni ellenanyag szintjének változását. Eredmények: A felmérésben részt vevo 295 dolgozó közül az oltást megelozoen 36 dolgozó esett át COVID-19-fertozésen (COVID-19-pozitív csoport). A második oltás után a megfigyelési idoszak három hónapjában COVID-19-fertozés nem alakult ki a felmérésben részt vevo oltott dolgozók körében. Az oltási reakciók enyhék voltak. A COVID-19-pozitív csoportban az N-antitestek medián küszöbértékindexe az elso vakcina után 4 héttel mérve szignifikánsan magasabb volt (28,37), mint a COVID-19-negatív (0,085) csoportban (p<0,0001). Az elso vakcina után 4 héttel az S-antitestek medián értéke (8015 U/ml) a COVID-19-pozitív csoportban szignifikánsan magasabb volt (p<0,0001), mint a COVID-19-negatív csoportban (23,18 U/ml). A COVID-19-negatív csoport S-antitest-középértéke a második vakcina után szignifikáns (p<0,0001), mintegy 500×-os emelkedést mutatott (23,18 U/ml rol 1173 U/ml-re). Egy vakcina hatásosságát a fertozések terjedésének megakadályozása igazolja. Következtetések: A második vakcina utáni megfigyelési idoszakban új COVID-19-fertozés nem volt az oltott dolgozók körében. A fertozésen át nem esett COVID-19-negatív egyének esetén az S-antitest emelkedése mérsékelt az elso oltás után, míg a második oltás után lényegesen emelkedik. A COVID-19-fertozésen átesett egyének csoportjában már az elso vakcina is jelentos S-antitest-termelodést vált ki. Orv Hetil. 2021; 162(39): 1551-1557. INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic caused global public health and economic crises. OBJECTIVE: The aim of this observation study was to estimate the effectiveness, safety and elicited immune response of the BNT162b2 mRNA Pfizer-BioNTech vaccine in healthcare workers of the Buda Hospital of the Hospitaller Order of St. John of God. METHOD: After vaccination, the infection rate, adverse events and the kinetics of anti-SARS-CoV-2 spike (S) protein and anti-SARS-CoV-2 nucleocapsid (N) protein antibodies were evaluated. RESULTS: Before vaccination, from the 295 healthcare workers 36 recovered from prior COVID-19 infection (COVID-19-positive group). After the second vaccination, there was no COVID-19 infection during the three-month follow-up period. The adverse events were mild. In the COVID-19-positive group, the median cut-off index of anti-N antibodies measured at 4 weeks after the first vaccination were significantly (p<0.0001) higher (28.37) than in the COVID-19-negative group (0.085). After the first vaccine, the median titer of anti-S antibodies was significantly higher (p<0.0001) in the COVID-19-positive group (8015 U/ml) compared to the COVID-19-negative group (23.18 U/ml). In the COVID-19-negative group, the median titer of anti-S antibodies increased significantly (p<0.0001) after the second vaccine (from 23.18 U/ml to 1173 U/ml), showing an increase of 500×. CONCLUSIONS: After the second vaccination, there was no COVID-19 infection during the follow-up. In the COVID-19-negative group, the anti-S antibody titer is moderate after the first vaccination and increases significantly after the second vaccine. In the COVID-19-positive group, the first vaccine induces significant anti-S antibody production. Orv Hetil. 2021; 162(39): 1551-1557.

Treatment Satisfaction, Patient Preferences, and the Impact of Suboptimal Disease Control in a Large International Rheumatoid Arthritis Cohort: SENSE Study.

BACKGROUND: Patients' needs and perspectives are important determinants of treatment success in rheumatoid arthritis (RA). Assessing patients' perspectives can help identify unmet needs and enhance the understanding of treatment benefits. OBJECTIVE: The SENSE study assessed the impact of inadequate response to disease-modifying antirheumatic drugs (DMARDs) on treatment satisfaction, disease outcomes, and patient perspectives related to RA disease management. METHODS: SENSE was a noninterventional, cross-sectional study conducted in 18 countries across Europe, Asia, and South America. Adult patients with poorly controlled RA of moderate/high disease activity were eligible. Patient satisfaction was assessed by the Treatment Satisfaction Questionnaire for Medication (TSQM v1.4). Treatment adherence, healthcare resource utilization (HRU), quality of life (QoL), work ability, digital health literacy (DHL), patient preference information, and treatment strategy were also assessed. RESULTS: A total of 1624 patients were included in the study: most were female (84.2%) and middle-aged, and mean disease duration was 10.5 years. Mean TSQM global satisfaction subscore was 60.9, with only 13.5% of patients reporting good treatment satisfaction (TSQM global ≥80). The strongest predictor of good treatment satisfaction was treatment with advanced therapies. Most patients (87.4%) reported good treatment adherence. In general, patients had impaired QoL and work ability, high HRU, and 67.4% had poor DHL. Leading treatment expectations were "general improvement of arthritis" and "less joint pain". Most patients preferred oral RA medications (60.7%) and rapid (≤1 week) onset of action (71.1%). "Increased risk for malignancies" and "increased risk for cardiovascular disease" were the least acceptable side effects. Despite suboptimal control, advanced therapies were only used in a minority of patients, and DMARD switches were planned for only half of the patients. CONCLUSION: Suboptimal disease control negatively impacts treatment satisfaction, work ability, QoL, and HRU. Data collected on patient perspectives may inform shared decision-making and optimize treat-to-target strategies for improving patient outcomes in RA.

Patients with rheumatoid arthritis facing sick leave or work disability meet varying regulations: a study among rheumatologists and patients from 44 European countries.

OBJECTIVES: To describe and explore differences in formal regulations around sick leave and work disability (WD) for patients with rheumatoid arthritis (RA), as well as perceptions by rheumatologists and patients on the system's performance, across European countries. METHODS: We conducted three cross-sectional surveys in 50 European countries: one on work (re-)integration and social security (SS) system arrangements in case of sick leave and long-term WD due to RA (one rheumatologist per country), and two among approximately 15 rheumatologists and 15 patients per country on perceptions regarding SS arrangements on work participation. Differences in regulations and perceptions were compared across categories defined by gross domestic product (GDP), type of social welfare regime, European Union (EU) membership and country RA WD rates. RESULTS: Forty-four (88%) countries provided data on regulations, 33 (75%) on perceptions of rheumatologists (n=539) and 34 (77%) on perceptions of patients (n=719). While large variation was observed across all regulations across countries, no relationship was found between most of regulations or income compensation and GDP, type of SS system or rates of WD. Regarding perceptions, rheumatologists in high GDP and EU-member countries felt less confident in their role in the decision process towards WD (ß=-0.5 (95% CI -0.9 to -0.2) and ß=-0.5 (95% CI -1.0 to -0.1), respectively). The Scandinavian and Bismarckian system scored best on patients' and rheumatologists' perceptions of regulations and system performance. CONCLUSIONS: There is large heterogeneity in rules and regulations of SS systems across Europe in relation to WD of patients with RA, and it cannot be explained by existing welfare regimes, EU membership or country's wealth.

Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis.

BACKGROUND: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. METHODS: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. RESULTS: KD values of affinity-purified ACPA IgGs varied between 10-6 and 10-8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two-two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. CONCLUSIONS: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

Regulatory B cells in rheumatoid arthritis: Alterations in patients receiving anti-TNF therapy.

Cytokines, including tumor necrosis factor alpha (TNFα) are involved in Rheumatoid arthritis (RA) pathogenesis by augmenting autoimmunity, sustaining long term inflammation in the synovium, and promoting joint damage. Anti-TNF therapy is one of the most efficient and widely used therapies for RA, although its mechanism is not clarified yet. Earlier we demonstrated that RA patients have a reduced number of IL-10 producing regulatory B cells (B10 cells) as compared to healthy individuals and they are functionally impaired. Our aim was to study the influence of anti-TNF therapy on B10 cells in RA, to follow the alteration of B cell activation markers (CD25, CD69) and to monitor the level of citrullinated peptid-specific antibodies and the secreted IL-10 in patients' sera during the therapy. We have observed that at six month after starting the therapy the frequency of B10 cells remarkably increased, while the expression of the activation marker, CD69 decreased on B cells. In contrast, serum levels of IL-10 and anti-citrullinated peptide antibodies did not change post-treatment. CONCLUSION: The reduced activation state of B cells and the increasing number of regulatory B10. cells might contribute to the therapeutic efficacy of anti-TNF agents in RA.

Induction and Differentiation of IL-10-Producing Regulatory B Cells from Healthy Blood Donors and Rheumatoid Arthritis Patients.

The most important feature of B cells is the production of Abs upon activation; additionally, B cells produce pro- and anti-inflammatory cytokines in response to certain stimuli. IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) that suppress autoimmune and inflammatory responses. B cells play a crucial role in the development and maintenance of the chronic inflammatory autoimmune disease rheumatoid arthritis (RA); however, controversial data are available on IL-10- producing Bregs in RA. Our aim was to identify the optimal conditions that induce IL-10+ Bregs and, furthermore, to shed light on the signaling pathways that are responsible for their expansion. The results show that dual stimulation by CpG and CD40L for 48 h is optimal for IL-10 induction, and this can be synergistically boosted by IL-21. We identified the CD19+CD27+ memory B cell population as the major source of IL-10+ Bregs. We detected significantly fewer CD19+CD27+IL-10+ cells in RA patients compared with healthy controls, and these were functionally defective in suppressing IFN-γ production by CD4+ T cells in coculture. IL-21 drastically increased the number of IL-10+ Bregs within the CD19+CD27+ and CD19+CD27- populations; furthermore, it induced the appearance of IL-10+Blimp-1+ plasmablasts. Monitoring the phosphorylation of key signaling molecules revealed that activation of ERK, p38, and CREB is indispensable for the induction of IL-10 production, whereas phosphorylation of STAT3 further enhances IL-10 expression in human Bregs. We conclude that CREB and STAT3 are the key transcription factors responsible for the expansion and differentiation of human IL-10-producing Bregs.

In vitro eradication of citrullinated protein specific B-lymphocytes of rheumatoid arthritis patients by targeted bifunctional nanoparticles.

BACKGROUND: Autoreactive B cells are crucial players in the pathogenesis of rheumatoid arthritis (RA). Autoantibodies specific for citrullinated proteins (ACPA), present in the serum of approximately 60-70 % of patients, have a pathogenic role in the disease. B cell depleting therapies may result in a transient immunosuppression, increasing the risk of infections. Our aim was to develop a new therapeutic approach to selectively deplete the ACPA producing autoreactive B cells. METHODS: To target B cells synthetic citrullinated peptide derived from the ß chain of fibrin, ß60-74Cit 60,72,74 (ß60-74Cit), the predominant epitope recognized by ACPA was used. Complement dependent cytotoxicity (CDC) was induced by a modified peptide derived from gp120 of HIV-1. To trigger CDC both the targeting peptide and the complement activating peptide were covalently coupled in multiple copies to the surface of poly (DL-lactic-co-glycolic acid) nanoparticles (NPs). Ex vivo antibody synthesis was examined by ELISA and ELISpot. CDC was tested after dead cell staining by flow cytometry. RESULTS: The ß60-74Cit peptide was selectively recognized by a small subset of B cells from RA patients having high level of peptide specific serum antibody, suggesting that the peptide can target diseased B cells. The modified gp120 peptide covalently coupled to NPs induced the formation of the complement membrane attack complex, C5b-9 in human serum. We show here for the first time that bifunctional NPs coupled to multiple copies of both the targeting peptide and the complement activating effector peptide on their surface significantly reduce ß60-74Cit peptide specific ex vivo ACPA production, by inducing complement dependent lysis of the citrullinated peptide specific B cells of seropositive RA patients. CONCLUSIONS: Bifunctional NPs covalently coupled to autoantigen epitope peptide and to a lytic peptide activating complement may specifically target and deplete the peptide specific autoreactive B-cells.

The EULAR Study Group for Registers and Observational Drug Studies: comparability of the patient case mix in the European biologic disease modifying anti-rheumatic drug registers.

OBJECTIVE: Under the auspices of the European League Against Rheumatism (EULAR), a study group of investigators representing European biologic DMARD (bDMARD) registers was convened. The purpose of this initial assessment was to collect and compare a cross section of patient characteristics and collate information on the availability of potential confounders within these registers. METHODS: Baseline characteristics of patients starting their first bDMARD in an arbitrary year (2008) for the treatment of RA, including demographic and disease characteristics, bDMARD drug details and co-morbidities, were collected and compared across 14 European bDMARD registers. RESULTS: A total of 5320 patients were included. Half the registers had restricted recruitment to certain bDMARDs during the study year. All registers` collected data on age, gender, disease duration, seropositivity for IgM-RF and 28-joint DAS (DAS28). The mean DAS28 ranged from 4.2 to 6.6 and the mean HAQ from 0.8 to 1.9. Current smoking ranged from 9% to 34%. Nine registers reported co-morbidities with varying prevalence. CONCLUSION: In addition to demonstrating European-wide collaboration across rheumatology bDMARD registers, this assessment identified differences in prescribing patterns, recruitment strategies and data items collected. These differences need to be considered when applying strategies for combined analysis. The lack of a common data model across Europe calls for further work to harmonize data collection across registers.

Bead arrays for antibody and complement profiling reveal joint contribution of antibody isotypes to C3 deposition.

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen ß and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen ß peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.

TGFß activated kinase 1 (TAK1) at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR), receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R) and the innate receptor, Toll-like receptor 9 (TLR9). However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF) and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs), ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1) is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

Acceptability of less than perfect health states in rheumatoid arthritis: the patients' perspective.

Some health problems are considered by many individuals as a 'normal' part of ageing. Our aim was to investigate whether patients with rheumatoid arthritis (RA) consider different types and levels of health losses as acceptable beyond a certain age. A multicenter cross-sectional survey was performed involving RA patients at the initiation of the first biological therapy. The EQ-5D and the Health Assessment Questionnaire Disability Index (HAQ-DI) questionnaires were used to describe domain-specific health states. Patients were asked to indicate for each domain from what age and onward (between ages 30 and 80 years in 10 year intervals) they considered moderate and severe problems acceptable or alternatively never acceptable. Seventy-seven RA patients (females 86%, mean age 50.3, disease duration 9.1 years) completed the questionnaire. Disease activity (DAS28), EQ-5D and HAQ-DI scores were mean 6.00 (SD 0.85), 0.35 (SD 0.36), 1.48 (SD 0.66), respectively. The majority of the patients considered age 70 and beyond as acceptable to have some health problems (EQ-5D: self-care 42%, pain/discomfort 34%, mobility 33%, usual activities 33%, anxiety/depression 27%), whilst at ages 30 and 40 as not acceptable. Severe health problems were mostly (57-69%) considered never acceptable, except the 'Usual activities' domain (acceptable from age 80 by 50.6%). The great majority of the patients (77-96%) were younger than what they indicated as the acceptability age limit. Similar results were found for the HAQ-DI. This small experimental study suggests that RA patients consider some health problems acceptable. This acceptability is age related and varies by health areas. Further larger studies are needed to explore explanatory variables and to compare with other diseases. Owing to the impact acceptability might have on RA patients' self-evaluation of current health state and decision-making, the topic deserves methodological improvement and further investigation.

Subjective health expectations at biological therapy initiation: a survey of rheumatoid arthritis patients and rheumatologists.

Subjective health expectations of patients with rheumatoid arthritis (RA) and rheumatologists remain understudied. We measured subjective expectations regarding treatment effects of biologicals as well as future length and quality of life. Moreover, we compared expectations regarding treatment effects to actual treatment effects. We recruited a sample of Hungarian RA patients initiating treatment with biologicals. Expectations regarding treatment effects and future health were obtained through a written questionnaire from patients and physicians, including functional impairment (HAQ-DI), health status (EQ-5D) and disease activity (DAS28). After three months, actual treatment effects were obtained. Ninety-two RA patients (females N = 81, 88%) with mean age of 51 (SD 12) and disease duration of 9 (SD 8) years with high average disease activity (DAS28: 6.1) were included. Patients expected significant and large health improvement within three months with mean changes on the HAQ-DI of -0.8 and on the EQ-5D of +0.4. Rheumatologists' estimates were similar, and they expected significant decrease of 2.3 on the DAS28. Actual scores after three months were obtained for 77 patients. The measured scores were significantly lower than expected scores for the HAQ-DI and EQ-5D. Rheumatologists' expectations for the DAS28 score were not significantly different from measured scores. Patients' average expectations regarding quality of life scores for ages 60, 70, 80 and 90 were 0.44, 0.24, 0.06 and -0.02, respectively. Our results suggest that both RA patients and rheumatologists expect quick and significant health improvements from biological drugs and tend to overestimate actual short term treatment effects. Nonetheless, RA patients expect a sharp deterioration of future health.

Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients.

Anti-citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline- and arginine-containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline-peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline-peptides identify antibody-secreting cells in in vitro cultures of RA B cells. Recognition of citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide-specific microarray. B cells were purified from blood by negative selection; antibody-producing cells were enumerated by ELISPOT assay. The panel composed of citrulline-peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline-peptide panel including the new short epitope peptide of filaggrin, fil311-315, also identified nearly one-third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide-specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline-containing filaggrin peptides (fil311-315 and fil306-326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline-peptides of filaggrin and vimentin detect ACPA-producing cells, and so could also be applied to study the B cells of RA patients.

Safety, pharmacokinetics, and biologic activity of pateclizumab, a novel monoclonal antibody targeting lymphotoxin α: results of a phase I randomized, placebo-controlled trial.

INTRODUCTION: Pateclizumab (MLTA3698A) is a humanized mAb against lymphotoxin α (LTα), a transiently expressed cytokine on activated B and T cells (Th1, Th17), which are implicated in rheumatoid arthritis (RA) pathogenesis. This study was conducted to assess the safety, tolerability, < NOTE: For clarity and per AMA/S-W Style, please restore the use of Oxford/serial commas (ie: David likes vanilla, strawberry, and chocolate ice cream) throughout. and biologic activity of single and multiple doses of intravenous (IV) or subcutaneous (SC) pateclizumab in RA patients. METHODS: The single ascending dose (SAD) phase in patients with stable RA consisted of six cohorts (4:1 active:placebo at 0.3 mg/kg IV, 1.0 mg/kg IV, 1.0 mg/kg SC, 3.0 mg/kg IV, 3.0 mg/kg SC, and 5.0 mg/kg IV; n = 5/cohort). In the multiple ascending dose (MAD) phase, patients with prespecified RA disease activity received three doses of pateclizumab or placebo (4:1) every 2 weeks (1.0 mg/kg SC, n = 10; 3.0 mg/kg SC, n = 20; or 5.0 mg/kg IV, n = 5). Safety and tolerability were assessed throughout, and clinical activity was determined after three doses (Week 6). RESULTS: We observed no serious adverse events (AEs) or dose-limiting toxicities, and the majority of AEs were mild to moderate. The pharmacokinetic profiles were linear, and clearance was independent of dose. Reductions in levels of serum CXCL13 were observed, supporting the biologic activity of pateclizumab on the LTα pathway. Patients receiving pateclizumab in the 3.0 mg/kg MAD group (3.0 mg/kg SC) demonstrated ACR20, ACR50, and ACR70 response rates at week 6 of 75%, 56% and 25%, respectively, compared with 57%, 29%, and 0% in the placebo group. The median Disease Activity Score in 28 joints, C-reactive protein, reduction was 28% for pateclizumab, versus 8.4% for placebo. CONCLUSIONS: Pateclizumabwas generally well-tolerated in RA patients. Preliminary evidence of clinical activity was observed in active RA patients at the dose level targeted for clinical effect.

Health-related quality of life and utility in patients receiving biological and non-biological treatments in rheumatoid arthritis.

Biological treatments earn increasing significance in the treatment of rheumatoid arthritis (RA) but are associated with high incremental cost-effectiveness ratio compared to conventional antirheumatic treatments such as disease-modifying antirheumatic drugs. As the most important objective of medical technologies should be to increase life years and/or patients' health-related quality of life (HRQoL), measuring QoL and utility in RA patients treated with biological therapies is crucial. The objective of this study is to compare the utility and QoL of patients treated with biological (n = 85) and non-biological (n = 168) antirheumatic drugs in Hungary in a cross-sectional non-interventional study. A measure of impairment (Disease Activity Score (DAS)-28), QoL measure (EuroQol five Dimension (EQ-5D) Visual Analogue Scale (VAS), Rheumatoid Arthritis Quality of Life (RAQoL)) and utility measures (indirect: EQ-5D index, direct: time trade-off (TTO)) were applied using an interview method. The Pearson correlation was used to assess the strength of the relationship of different measures in the total study group (n = 253). The EQ-5D index (biological treatment: 0.608, non-biological treatment: 0.483; P = 0.012) and DAS-28 (biological treatment: 3.8, non-biological treatment: 4.5; P = 0.003) showed statistically significant difference between the two subcohorts after adjusting data by age, gender and disease duration. Our results indicate that patients on biological treatment have lower disease activity and higher utility; however, it was not statistically significant in all cases. According to our knowledge, TTO was not used previously in Hungarian RA patients. Utility data concerning biological treatments are essential for cost-utility models in health technology assessment reports for public reimbursement.

[The assessment of oral NSAID use in patients with rheumatoid arthritis in Hungary--a cross sectional non interventional study]. / Orális nem-szteroid gyulladásgátló gyógyszerek felhasználásának vizsgálata a Budai Irgalmasrendi Kórház. II. Reumatológia Osztályának rheumatoid arthritises betegei között.

Continuous NSAID (nonsteroidal anti-inflammatory drug) therapy is associated with gastrointestinal (GI) and cardiovascular (CV) side effects. In this paper, the oral NSAID use of 143 patients with rheumatoid arthritis was assessed focusing on safety and farmacoeconomic aspects in a cross sectional non interventional study. The most widely used NSAIDs were meloxicam (n = 55, 38.5%) and diclofenac (n = 30, 21%). We found that coxibs were overused (n = 13, 9.1%) compared with the average total coxib consumption in Hungary. According to our results, drugs associated with GI friend side effect profile (meloxicam, celecoxib, etoricoxib) were much preferred in patients with previous GI events, than in patients with low GI risk. The previous occurrence of GI events were significantly higher (p = 0.019) in patients currently treated with safer NSAIDs, probably because of the so-called 'indication bias'. No statistically significant difference in patient's quality of life could be proved between NSAID drug groups. The uses of NSAIDs were considered to be rational concerning CV and GI risk as well as cost-effectiveness.