Plasma Of Rheumatoid Arthritis Patients Research Articles

Gelsolin alleviates rheumatoid arthritis by negatively regulating NLRP3 inflammasome activation

Despite numerous biomarkers being proposed for rheumatoid arthritis (RA), a gap remains in our understanding of their mechanisms of action. In this study, we discovered a novel role for gelsolin (GSN), an actin-binding protein whose levels are notably reduced in the plasma of RA patients. We elucidated that GSN is a key regulator of NLRP3 inflammasome activation in macrophages, providing a plausible explanation for the decreased secretion of GSN in RA patients. We found that GSN interacts with NLRP3 in LPS-primed macrophages, hence modulating the formation of the NLRP3 inflammasome complex. Reducing GSN expression significantly enhanced NLRP3 inflammasome activation. GSN impeded NLRP3 translocation to the mitochondria; it contributed to the maintenance of intracellular calcium equilibrium and mitochondrial stability. This maintenance is crucial for controlling the inflammatory response associated with RA. Furthermore, the exacerbation of arthritic symptoms in GSN-deficient mice indicates the potential of GSN as both a diagnostic biomarker and a therapeutic target. Moreover, not limited to RA models, GSN has demonstrated a protective function in diverse disease models associated with the NLRP3 inflammasome. Myeloid cell-specific GSN-knockout mice exhibited aggravated inflammatory responses in models of MSU-induced peritonitis, folic acid-induced acute tubular necrosis, and LPS-induced sepsis. These findings suggest novel therapeutic approaches that modulate GSN activity, offering promise for more effective management of RA and a broader spectrum of inflammatory conditions.

Level of glial cell derived neurotrophic factor in the blood plasma of rheumatoid arthritis patients and its relationship with alexithymia.

Glial cell derived neurotrophic factor (GDNF) has an important role in the pathogenetic mechanisms and clinical manifestations of rheumatoid arthritis (RA). Alexithymia is associated with a severe clinical course and worse prognosis, while the relationship between alexithymia and GDNF in RA patients has not been investigated before. The aims of the study were to investigate the GDNF level in blood plasma in RA patients depending on the presence of alexithymia and to evaluate the relationship of GDNF level with clinical manifestation and quality of life. Fifteen men and 73 women with RA were examined using the Disease Activity Score with 28-joint count (DAS28) with erythrocyte sedimentation rate (ESR) index, the Simple Disease Activity Index (SDAI), the Rheumatoid Arthritis Clinical Disease Activity Index (CDAI), the Visual Analogue Scale (according to the assessment of the patient - VAS-P and the assessment of the doctor - VAS-D), the Health Assessment Questionnaire (HAQ), the Toronto Alexithymia Scale (TAS-20), the Disability Rating Index (DRI) and SF-36 indexes. Glial cell derived neurotrophic factor level in the blood plasma was determined by enzyme-linked immunosorbent assay (ELISA). Forty percent of RA patients had alexithymia. Glial cell derived neurotrophic factor level in the examined patients was 3.73 ±2.59 pg/ml, in patients with alexithymia 4.08 ±2.87 pg/ml, without alexithymia 3.48 ±2.37 pg/ml (p = 0.295). Patients with alexithymia had a higher erythrocyte sedimentation rate (ESR) and index scores than patients without alexithymia - ESR: 34.29 ±14.22 vs. 22.73 ±12.03 mm/h (p = 0.017), DAS28: 6.53 ±0.66 vs. 6.09 ±0.55 (p = 0.017), VAS-D: 7.19 ±0.81 vs. 6.53 ±0.83 (p = 0.020), HAQ: 1.78 ±0.58 vs. 1.51 ±0.54 (p = 0.040). Also they had worse SF-36 indicators - physical functioning: 39.52 ±13.78 vs. 51.00 ±14.90 (p = 0.019), role functioning due to physical condition: 30.95 ±20.77 vs. 46.67 ±24.76 (p = 0.041), physical component of health: 31.47 ±11.44 vs. 41.61 ±15.88 (p = 0.028). In patients with alexithymia, a correlation was found between the GDNF level and severity of pain according to VAS-P: rS = 0.338, p = 0.044, and VAS-D: rS = 0.446, p = 0.006. Alexithymia was found in 40% of RA patients. Rheumatoid arthritis patients with alexithymia had a nonsignificantly higher GDNF level compared to patients without alexithymia. In RA patients with alexithymia, an association of GDNF level in the blood plasma with RA activity, loss of functional capacity and reduced quality of life was established. Alexithymia in RA patients is an important factor in the clinical manifestation of RA and modification of the pathophysiological role of GDNF.

Inhibition of Oxidative Stress-Induced Ferroptosis Can Alleviate Rheumatoid Arthritis in Human.

Rheumatoid arthritis (RA) is a chronic autoimmunity illness, mainly featured with synovitis of the joint. The specificity of ferroptosis is disparate in different diseases, and the mechanism of ferroptosis in RA has some uncertainty yet. Therefore, the mechanism of ferroptosis was deeply observed in RA patients and animal models. In this paper, plasma of RA patients, the tumor necrosis factor-alpha-induced human synovial fibroblasts, and an animal model of arthritis induced by collagen were applied to initially inquire about the therapeutic effect of ferroptosis. For the RA patients, ELISA detected protein expression of glutathione (GSH), GPX4, Nrf2, Keap-1, and ferritin. In cell experiments, erastin or fer-1 regulated the invasion of human synovial fibroblast cells, mitochondrial membrane potential, reactive oxygen species (ROS) expression, marker protein, and so on. For the animal experiments, 32 Sprague-Dawley male rats were randomly separated into four groups with a collagen-induced RA model for 14 days and administered with erastin or fer-1 for 35 days. The expressions of GSH, GPX4, Nrf2, and Keap-1 were lower, and the ferritin was higher in RA patients, and the expressions of these proteins varied significantly after disease remission. In addition, ferroptosis inactivation also reduced the proliferation and migration ability, mitochondrial membrane potential, and ROS in cells. We discovered unexpectedly that activation of ferroptosis meaningfully forbore the foot swelling in animals with CIA, reduced arthritis scores, destruction of bone, and articular synovitis, and also decreased the high expression of inflammatory factors in plasma. There is a nonlinear relationship between human disease manifestations and animal model pathology. Ferroptosis regulating in RA for humans or animals may produce different effects.

Specific Increase in Joint Neutrophil Extracellular Traps and Its Relation to Interleukin 6 in Autoimmune Arthritis.

Neutrophils and their extracellular traps have been shown to play an important role in the pathogenesis of rheumatoid arthritis (RA), but the detailed mechanisms in joints are still unclear, and their regulation remains to be solved. Here, we explored neutrophil extracellular trap (NET)osis in experimental models of arthritis and further investigated the effects of interleukin-6 (IL-6) inhibition in neutrophils and NETosis. In skins of peptide GPI-induced arthritis (pGIA), citrullinated protein was detected as well as citrullinated histone expression in immunized skin but this was not specific to pGIA. Citrullinated histone expression in pGIA joints was specific to pGIA and was merged with neutrophil elastase, suggesting NETosis. Neutrophils in joints tend to upregulate IL-6 receptors when compared with bone marrow neutrophils. Administration of mouse anti-IL-6 receptor antibodies in pGIA suppressed arthritis in association with a decrease in neutrophil infiltration and NETosis in joints. In the plasma of RA patients, citrullinated protein was significantly reduced after tocilizumab treatment. Our results suggest that IL-6 enhances neutrophil chemotaxis and NETosis in inflammatory joints and could be the source of citrullinated proteins.

Plasma Levels of MicroRNA-146a-5p, MicroRNA-24-3p, and MicroRNA-125a-5p as Potential Diagnostic Biomarkers for Rheumatoid Arthris.

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by inflammation of the articular tissue. This study aims to evaluate the expression of microRNA (miR)-146a-5p, miR-24-3p, and miR-125a-5p in the plasma of RA patients and compare them with those of healthy controls to obtain a specific expression profile for earlier diagnosis and assistance in treating patients. This study was performed on 50 RA patients and 50 healthy controls. Five microliters of blood were taken from each patient/control. Plasma RNA was extracted using the Trisol solution. cDNAs were synthesized; using moloney murine leukemia virus (MMLV) and deoxynucleoside triphosphate (dNTP). Real-time PCR was performed using SYBR green kit. The mean expression of miR-146a-5p, miR-24-3p, and miR-125a-5p in the RA group were 8.1±1.9, 6.5±1.2, and 6.8±2.2 and in the healthy group were 4.8±1.6, 3.6±2.2, and 3.4±1.7, respectively. Significant differences were also observed in the mean expression of these three miRNAs in four subgroups of RA patients with different disease activity based on disease activity score 28 (DAS28) (p<0.05). ROC curve analysis showed that miR-146a-5p (AUC=0.8, sensitivity= 96%, specificity=86%), miR-24-3p (AUC=0.7, Sensitivity=95%, Specificity=75%) and miR-125a-5p (AUC=0.71, sensitivity=93%, specificity=84%) could be used as suitable biomarkers for RA diagnosis. Increased expressions of miR-146a-5p, miR-24-3p, and miR-125a-5p in RA patients indicate that the miRNAs are involved in disease incidence and progression, and the measurement of their expression can play an essential role in the diagnosis and treatment of the disease.

AB0061 ALTERATIONS OF XANTHINE OXIDOREDUCTASE ACTIVITY IN RED BLOOD CELLS AFTER GLUCOCORTICOID TREATMENT IN RHEUMATOID ARTHRITIS

Background:According to modern concepts, rheumatoid arthritis (RA) refers to severe autoimmune rheumatic diseases. The activation of free radical oxidation processes is essential in the development of this disease [1]. Xanthine oxidoreductase is a significant reactive oxygen species source [2]. Despite the great advances in the treatment of rheumatoid arthritis (RA) associated with the introduction of innovative drugs and especially the improvement of the strategy for their use into clinical practice, glucocorticoids still remain an important component of RA pharmacotherapy in actual clinical practice.Objectives:to evaluate the changes in activities of xanthine oxidoreductase interconvertible forms (xanthine oxidase, ЕС 1.17.3.2 and xanthine dehydrogenase, ЕС 1.17.1.4) in lysed red blood cells of RA patients in relation with glucocorticoid treatment.Methods:47 RA patients with verified RA and 30 healthy controls were enrolled in the study. The diagnosis was verified using the 2010 ACR/EULAR criteria 2010. All patients have moderate DAS28 scores. RA patients were randomized into 2 groups comparable in gender, age and the principal clinical manifestations. Methylprednisolone (Metipred, Orion Corp.), average dose 30 mg/day, and betamethasone (Diprospan, Schering-Plough), single dose7 mg, were administered intramuscularly in the respective groups. Хanthine oxidase (XO) and xanthine dehydrogenase (XDG) activities were measured in lysed red blood cells by spectrophotometric method as previously described [3]. The changes of these enzymes activities were studied in RA patients before and after the injection of glucocorticoids. Statistical comparison tests were selected in according to common guidelines, differences were considered significant when p&lt;0.05. Central tendencies were expressed as means±SEM.Results:Mean age of patients in methylprednisolone group was 41.8±1.05 years, and mean RA duration (± SEM) was 7.9±0.21 years. Mean age of patients in diprospan group was 40.9±1.07 years, and mean RA duration was 8.0±0.33 years. Significant decreases of XO activity and increase of XDG activity were observed in lysed red blood cells of RA patients just after the injection of each glucocorticoid drug. Changes of the enzymatic activities in lysed red blood cells were more pronounced in methylprednisolone group. However enzymatic activity did not reach the level of healthy controls. As described previously, decreased XO activity and increased XDG activity were observed in plasma of RA patients just after the injection of the average therapeutic doses of glucocorticoids, as well as in lysed lymphocytes just after the injection of methylprednisolone [4].Conclusion:Treatment with methylprednisolone and betamethasone can affect the balance of XO/XDG activity and increase the antioxidant potential of the blood. This effect can exert beneficial influence on autoimmune inflammation in RA.

Increased Extracellular ATP in Plasma of Rheumatoid Arthritis Patients Activates CD8+T Cells

Rheumatoid arthritis (RA) is an autoimmune disorder with genetic and environmental causes often linked with the disease etiology. A disrupted metabolism has often been a characteristic of RA and an altered metabolic state of immune cells has been associated with their phenotypic and functional changes. The energy in the form of ATP produced by the metabolically active cells may thus initiate a cascade of immune responses there by influencing the disease pathogenesis or progression. Through this study we have focused on determining the role of ATP in etiology of RA and aberrant cellular functions. Blood samples of 80 healthy controls (HC) and 95 RA patients were screened for extracellular ATP concentration, transcriptome analyses, an inflammatory mediator and the results were statistically analysed. In this study, ATP is shown to be excessive in the plasma of RA patients (453.5±16.09% in RA vs. 233.9±10.07% in HC, p<0.0001) and significantly increases with the disease severity. The abundant extracellular ATP could activate circulating cytotoxic CD8+T cells in RA patients to produce Granzyme B. Plasma ATP is thus identified to have a significant potential in progression and prognosis of RA and may thus be studied further to design better therapeutic approaches for the disease.

Activated Platelets Convert CD14+CD16- Into CD14+CD16+ Monocytes With Enhanced FcγR-Mediated Phagocytosis and Skewed M2 Polarization.

Monocytes are important cellular effectors of innate immune defense. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. The expansion of intermediate CD14+CD16+ monocytes has been reported in chronic inflammatory diseases including rheumatoid arthritis (RA). However, the mechanism underlying induction of CD16 and its role in monocytes remains poorly understood. Here, we demonstrate that activated platelets are important for induction of CD16 on classical CD14+CD16- monocytes by soluble factors such as cytokines. Cytokine neutralization and signaling inhibition assays reveal that sequential involvement of platelet-derived TGF-β and monocyte-derived IL-6 contribute to CD16 induction on CD14+CD16- monocytes. Activated platelet-induced CD16 on monocytes participates in antibody-dependent cellular phagocytosis (ADCP) and its level is positively correlated with phagocytic activity. CD14+CD16- monocytes treated with activated platelets preferentially differentiate into M2 macrophages, likely the M2c subset expressing CD163 and MerTK. Lastly, the amount of sCD62P, a marker of activated platelets, is significantly elevated in plasma of RA patients and positively correlates with clinical parameters of RA. Our findings suggest an important role of activated platelets in modulating phenotypical and functional features of human monocytes. This knowledge increases understanding of the immunological role of CD14+CD16+ cells in chronic inflammatory diseases.

Association of Toll-like Receptor 4 Rs7873784 G/C Polymorphism with Rheumatoid Arthritis Risk in a Chinese Population

ABSTRACT Background The purposes of this study were to explore whether the Toll-like receptor 4 (TLR4) gene rs7873784 G/C polymorphism was related to the risk of rheumatoid arthritis (RA) and to the clinical features of the disease in Chinese subjects. Materials and Methods We examined the TLR4 rs7873784 G/C polymorphism in 805 Chinese RA patients and 1095 healthy controls. Genotype was determined with a custom-by-design 48-Plex single nucleotide polymorphism scan™ Kit. Blood plasma levels of TLR4 in 170 RA patients and 170 matched controls were measured by ELISA. Results The TLR4 gene rs7873784 G/C polymorphism was related to a reduced risk for RA. By stratified analysis, we found a dramatically reduced risk for RA in patients who were female, CRP-positive, RF-positive, DAS28 ≥ 3.20, or ESR ≥ 25. Compared with the controls, the average level of TLR4 protein in plasma of RA patients was increased. In addition, RA patients exhibited higher levels of TLR4 mRNA than controls (P < .05). Conclusion These results demonstrate the TLR4 rs7873784 G/C polymorphism to relate to a decreased risk for RA in a Chinese population.

Identification and characterization of adipose tissue-derived human antibodies with “anti-self” specificity

Abstract We have previously shown that the human obese adipose tissue (AT) contributes to increased secretion of adipocyte-specific IgG antibodies in individuals with obesity. We have also shown that this occurs without any exogenous stimulation, because the ongoing process of cell death in the obese AT leads to the release of “self” antigens able to induce chronic stimulation of B cells. Here, we confirm and extend our initial observation on a different cohort of individuals, and we show that also the plasma of obese individuals is enriched in IgG antibodies with specificities for adipocyte-derived antigens. Adipocyte-specific IgG secreted in the obese AT are significantly correlated with those present in plasma. Using immunoprecipitation and mass spectrometry, we have identified these antigenic specificities. The antigens are almost exclusively intracellular or cell-associated, usually not “self” antigens, but they are released by cells dying in the AT. These antigens will be used in protein arrays to screen plasma from individuals with obesity and also with autoimmune diseases. We have indeed preliminary evidence that the plasma of Rheumatoid Arthritis patients is enriched in adipocyte-specific IgG antibodies, suggesting that obesity may contribute to pathogenicity in autoimmune patients. Finally, we show for the first time that not only macrophages but also adipocytes in the obese AT are efficient antigen-presenting cells and stimulate the secretion of IgG autoimmune antibodies. They do so because they express the antigen-presenting molecules CD1d and, to a lesser extent, MHC class II, as well as the co-stimulatory molecule CD86. These results may lead to the development of novel therapeutic strategies to controlautoimmunity.

LncRNA CASC2 downregulation participates in rheumatoid arthritis, and CASC2 overexpression promotes the apoptosis of fibroblast‑like synoviocytes by downregulating IL‑17.

lncRNA cancer susceptibility candidate 2 (CASC2) is a recently identified oncogenic lncRNA in different types of cancers. Our preliminary microarray data showed that lncRNA CASC2 was downregulated in the plasma of patients with rheumatoid arthritis (RA), indicating the involvement of this lncRNA in RA. In the present study, lncRNA CASC2 and IL-17 in plasma were detected by reverse transcription--quantitative PCR and ELISA, respectively. Diagnostic analyses were performed using receiver operating characteristic curves. Flow cytometry was performed to evaluate cell apoptosis. The effects of lncRNA CASC2 on IL-17 expression were determined via western blotting. lncRNA CASC2 was found to be downregulated, while IL-17 was upregulated in the plasma of RA patients when compared with these levels in the plasma of healthy controls. Plasma levels of lncRNA CASC2 and IL-17 were significantly and inversely correlated in both RA patients and healthy controls. Altered plasma levels of lncRNA CASC2 and IL-17 were able to differentiate RA patients from healthy controls. Overexpression of lncRNA CASC2 promoted, while treatment with IL-17 inhibited the apoptosis of human fibroblast-like synoviocytes (HFLSs) isolated from RA patients. Overexpression of lncRNA CASC2 inhibited IL-17 expression in HFLS, while treatment with IL-17 did not significantly affect the expression of lncRNA CASC2. Therefore, downregulation of lncRNA CASC2 is involved in RA and lncRNA CASC2 overexpression may promote the apoptosis of HFLS by downregulating IL-17.

Activity of prooxidant and antioxidant systems enzymes in plasma of rheumatoid arthritis patients

Activity of prooxidant and antioxidant systems enzymes in plasma of rheumatoid arthritis patients

LncRNA GAS5 overexpression downregulates IL-18 and induces the apoptosis of fibroblast-like synoviocytes.

Long non-coding RNA (lncRNA) growth arrest specific transcript 5 (GAS5) negatively regulates interleukin-18 (IL-18) in ovarian cancer, while IL-18 contributes to the development of rheumatoid arthritis (RA). Therefore, GAS5 may also participate in RA. GAS5 and IL-18 in plasma of RA patients (n = 60) and healthy controls (n = 60) were measured by RT-qPCR and ELISA, respectively. Linear regression was performed to analyze the correlations between plasma levels of IL-18 and GAS5 in both RA patients and healthy controls. In the present study, we found that plasma GAS5 was downregulated, while IL-18 was upregulated in RA patients than in healthy controls. A significant and inverse correlation between GAS5 and IL-18 was found in RA patients but not in healthy controls. IL-18 treatment did not significantly alter the expression of GAS5 in fibroblast-like synoviocytes, while GAS5 overexpression led to the inhibited expression of IL-18. GAS5 overexpression also resulted in the promoted apoptosis of fibroblast-like synoviocytes. Therefore, GAS5 overexpression may improve RA by downregulating IL-18 and inducing the apoptosis of fibroblast-like synoviocytes. Key points • The present study mainly showed that overexpression of GAS5 may assist the treatment of RA. • The mechanism of GAS5 for the treatment of RA involves the downregulating inflammatory IL-18 and mediating the apoptosis of fibroblast-like synoviocytes. • GAS5 and IL-8 were correlated in RA patients but not in healthy controls.

Circulating microRNA-23b as a new biomarker for rheumatoid arthritis

MicroRNA-23b (miR-23b) is associated with inflammation and autoimmune diseases. This study evaluated miR-23b expression and assessed its potential as a biomarker of disease activity for rheumatoid arthritis (RA). Differential expression of microRNAs was determined by miRNA microarray analysis in fibroblast-like synoviocytes (FLSs) from four trauma patients as healthy controls (HCs) and eight RA patients. The microarray results showed elevated expression of miR-23b in FLSs from RA patients and this finding was corroborated by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization using synovial tissues (STs). Furthermore, we found miR-23b levels in plasma of RA patients were significantly higher than in HCs, and plasma miR-23b levels positively correlated with the erythrocyte sedimentation rate (ESR), hypersensitive C-reactive protein (hs-CRP), C-reactive protein (CRP), DAS28, and platelet (PLT) count (P < 0.05). MiR-23b levels in plasma inversely correlated with the levels of hemoglobin (Hb), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (P < 0.05), but not with rheumatoid factor (RF) or anti-cyclic citrullinated peptide antibodies (ACPA) (P > 0.05). Moreover, patients with anorexia showed higher levels of miR-23b in plasma than those without anorexia. Similar results were observed with fatigue. Appropriate treatment for RA not only ameliorated the disease condition but also reversed the elevated plasma miR-23b level remarkably. These results suggest that circulating miR-23b may be a promising biomarker for RA disease activity.

MicroRNA-766-3p Contributes to Anti-Inflammatory Responses through the Indirect Inhibition of NF-κB Signaling.

MicroRNA (miRNA) is small RNA of 20 to 22 nucleotides in length and is stably present in plasma. Regulating the expression of miRNA taken into cells has been suggested as a general therapeutic approach. We identified the novel anti-inflammatory miRNA hsa-miR-766-3p and investigated its biological function in human rheumatoid arthritis (RA) fibroblast-like synoviocyte MH7A cells. To verify the function of the miRNA present in the plasma of RA patients, we performed a comprehensive analysis of the miRNA expression during abatacept treatment and identified eight miRNAs with significantly altered expression levels. Among these eight miRNAs, miR-766-3p was found to have a clear function. The expression of inflammatory genes in response to inflammatory stimuli was suppressed in MH7A transduced with miR-766-3p. We showed that miR-766-3p indirectly reduced the activation of NF-κB and clarified that this mechanism was partially involved in the reduction of the mineralocorticoid receptor expression. In addition, the inflammatory responses were suppressed in other types of cells. These results indicate the novel function of miR-766-3p, findings that may aid in the development of therapies to suppress inflammation, not only in RA but also in other diseases.

Parvovirus B19 infection modulates the levels of cytokines in the plasma of rheumatoid arthritis patients

BackgroundParvovirus B19 (B19V) infection is associated with various autoimmune diseases. We investigated the levels of pro-inflammatory (IFNᵧ, TNFα, IL-2, IL-12) and anti-inflammatory (IL-4, IL-10) cytokines in the plasma of B19V DNA positive (B19+) and negative (B19−) rheumatoid arthritis (RA) patients in comparison with the control group (healthy persons). MethodsBlood samples were collected from 118 patients with RA and 49 healthy voluntaries. B19V sequence was determined in whole blood and cell-free plasma DNA by nested PCR. The levels of cytokines in the plasma and cell culture medium from Concanavalin A (ConA) or B19V VP1 protein stimulated PBMC were determined by ELISA. ResultsThe levels of IL-4, IL-10, IL-12, IL-2 and TNFα were higher in plasma of RA patients in comparison with control persons. B19+ controls and RA patients had lower levels of IFNᵧ in comparison with B19− controls and RA patients.Within RA patients the plasma levels of IFNᵧ were lower in patients with low RA disease activity or remission. Plasma level of IL-4 was increased and IL-10 level was decreased in B19+ RA patients in comparison with B19− RA patients and did not differ between B19+ and B19− controls. B19V infection did not affect plasma levels of IL-12, IL-2, and TNFα.ConA and B19 VP1 protein stimulated PBMC from RA patients produced less IFNᵧ than stimulated PBMC from the healthy controls. ConclusionsB19V infection could differently modulate the amount of cytokines in the plasma of healthy persons and RA patients. Decreased production of IFNᵧ and raised level of plasma IL-4 in RA patients could lower antiviral clearance.

High thioredoxin-1 levels in rheumatoid arthritis patients diminish binding and signalling of the monoclonal antibody Tregalizumab.

The humanized non-depleting anti-CD4 monoclonal antibody Tregalizumab (BT-061) is able to selectively activate the suppressive function of regulatory T cells and has been investigated up to phase IIb in clinical trials in patients suffering from rheumatoid arthritis (RA). A pharmacokinetic–pharmacodynamic model based on clinical data from RA and healthy volunteers, which used the cell surface CD4 downmodulation as marker of activity, confirmed a stronger effect in healthy volunteers compared with RA patients. We tried to understand this phenomenon and evaluated the influence of the small oxidoreductase thioredoxin-1 (Trx1). To counteract oxidative stress that is strongly associated with RA pathophysiology, the organism employs Trx1. Therefore, increased expression and secretion of Trx1 is found in the synovial fluid and plasma of RA patients. Moreover, the binding site of Tregalizumab is in close proximity to a disulphide bond in domain 2 (D2) of CD4, which is a known target for a reduction by oxidoreductase Trx1. With the experiments reported herein, we demonstrated that specific reduction of the D2 disulphide bond by Trx1 led to diminished binding of Tregalizumab to recombinant human soluble CD4 and membrane-bound CD4 on T cells. Moreover, we showed that this caused changes in the Tregalizumab-induced CD4 signalling pathway via the lymphocyte-specific protein tyrosine kinase p56Lck and CD4 downmodulation. In summary, we provide evidence that high Trx1 levels in RA patients compared with healthy subjects are a potential reason for diminished binding of Tregalizumab to CD4-positive T cells and offer an explanation for the observed decreased CD4 downmodulation in RA patients in comparison to healthy subjects.

The onset of lipid peroxidation in rheumatoid arthritis: consequences and monitoring

ABSTRACTSeveral epidemiological studies propose the association of rheumatoid arthritis (RA) with oxidative stress. The aim of this study was to estimate the possible onset of systemic lipid peroxidation in RA patients and its relevance for pathophysiology and monitoring of RA. Seventy-three patients with RA and 73 healthy subjects were included in the study. Lipid peroxidation was estimated by the measurement of 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal, malondialdehyde, acrolein, crotonaldehyde, 4-oxononenal, and isoprostanes (8-isoPGF2α) levels. Cytosolic phospholipase A2 (cPLA2), platelet-activating factor acetylhydrolase (PAF-AH) and glutathione peroxidase (GSH-Px) activities and vitamin E levels were also determined. In parallel, the plasma levels of phospholipid arachidonic acid (AA), linoleic acid (LA), and 4-HNE-protein adducts were monitored. Plasma of RA patients had increased vitamin E levels, but decreased GSH-Px activity and phospholipid AA and LA levels when compared to levels of the healthy subjects. The levels of aldehydes were significantly increased in the plasma of the RA patients and even more in urine. Significant increases in HNE-modified protein adducts was observed for the first time in plasma of RA patients, while the activities of PAF-AH and cPLA2 were decreased. The 8-isoPGF2α levels were 9-fold higher in plasma and 3-fold higher in urine of RA patients and were related to the severity of disease. The levels of lipid peroxidation products in plasma and in urine suggest the relationship between lipid peroxidation and the development of RA. Additionally, urine 8-isoPGF2α, plasma 4-HNE and 4-HNE-protein adducts appear to be convenient biomarkers to monitor progression of this autoimmune disease.

P84 - Lipid peroxidation in Rheumatoid arthritis; consequences and monitoring

The epidemiological studies have shown that rheumatoid arthritis (RA) leads to oxidative stress formation. Therefore the aim of this study has been to estimate the lipid peroxidation in RA patients. Moreover the reasons and consequences of changes in lipid peroxidation were estimated. 73 patients with RA and 62 healthy subjects were included into the study. Lipid peroxidation was estimated by the measurement of phospholipid arachidonic acid (AA) and linoleic acid (LA) as well as aldehydes: 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal (4-HHE), malondialdehyde (MDA), acrolein, crotonaldehyde, and 4-oxononenal (4-ONE) that were determined by GC and GCMS, while 8-isoprostanes (8-isoPGF2a) – was determined by LCMS. Phospholipase A2 (PLA2), platelet-activating factor acetylhydrolase (PAF-AH) and glutathione peroxidase (GSH-Px) activity were determined spectrophotometrically, while vitamin E was determined by HPLC. Plasma of RA patients was characterized by higher vitamin E level and decreased GSH-Px activity as well as the level of phospholipid AA and LA compared to healthy subjects. The level of all examined aldehydes was significantly increased in plasma patients but higher increase was observed in urine of RA patients in comparison with control group. The 8-isoprostanes level was approximately 9-fold higher in the plasma and 3-fold higher in the urine of RA patients compared to healthy subjects, but in the urine the changes in 8-isoprostanes level was correlated with RA progression. A significant increase in 4-HNE-modified protein adducts level as well as a decrease in the activity of PAF-AH and PLA2 were observed in the plasma of RA patients. The level of lipid peroxidation products in the plasma and the urine may be the indicator of RA, but additionally urine 8-isoprostanes may be the useful and reliable biomarker of RA progression.

Anticyclic citrullinated protein antibodies are implicated in the development of cardiovascular disease in rheumatoid arthritis.

Previous studies have suggested a relationship between anticyclic citrullinated protein (CCP) levels and development of cardiovascular disease in rheumatoid arthritis (RA). However, a limited number of studies have demonstrated an involvement of anti-CCPs in those processes. This study was aimed to define the specific role of these auto-antibodies in the pro-oxidative, inflammatory, and proatherogenic profile observed in leukocytes from RA patients. Seventy-five RA patients and 31 healthy donors were enrolled. Carotid intima media thickness was evaluated as atherosclerosis marker. Several procoagulant and inflammatory factors, leukocyte activation, and oxidative stress markers were analyzed in plasma and leukocyte subsets. Anti-CCPs were purified from plasma of RA patients, and in vitro treatment of healthy leukocytes was conducted. High titers of anti-CCPs were associated to altered expression of prothrombotic and inflammatory markers, high oxidative stress, and pathological carotid intima media thickness in RA patients. Notably, gene expression analysis showed that lymphocytes were major players in altered inflammatory profile, monocytes were responsible for the protrombotic and atherogenic status, and neutrophils mainly displayed a pro-oxidative feature. In vitro treatment with purified anti-CCPs fully recapitulated that pathogenic profile, promoting the activation of leukocytes. Anti-CCPs are key players in the inflammatory and proatherogenic status of RA patients. The effects are specific of the immune cell targeted, promoting overexpression of thrombotic, inflammatory, and pro-oxidative markers in monocytes; pro-oxidative status in neutrophils; and proinflammatory profile in lymphocytes. Targeting these autoantibodies would be an excellent strategy to prevent the development of cardiovascular disease in RA.