Background. Antinuclear antibodies (ANA) are a group of antibodies that target nuclear and cytoplasmic antigens. Testing for ANA using an immunofluorescence assay (IFA) on HЕp-2 cell (IFA-HEp-2) is a screening test for the diagnosis of systemic autoimmune rheumatic diseases, druginduced lupus erythematosus, autoimmune liver diseases, juvenile idiopathic arthritis. In routine ANA testing final report forms bears important, but unstandardized variables (e. g., screening and final titers of ANA, coding of glow types, the procedure for performing confirmatory tests). Aim of the study. Develop a standard report form for IFA-HEp-2 testing results.Material and methods. Survey of 10 immunological and clinical diagnostic laboratories using the ICAP questionnaire adapted by the Working Group on Standardization of the definition of ANA by the IFA-HEp-2 method (Committee on Immunology of the Association ‘Federation of Laboratory Medicine’).Results. According to the results of a survey: most of the participants use a screening dilution of serum 1:160; indicates the maximum end titer of antinuclear factor (ANF), cytoplasmic staining; identifies staining types (including AC encoding); considers it mandatory to make confirmatory tests to detect ANA to individual nuclear antigens in ANF-positive patients, but does not provide a list of this tests in the results form; describes ANF titer for each of the detected glows separately; designates ANF titers using a colon and does not give a clinical interpretation of the results. Based on interlaboratory consensus, ICAP recommendations and the requirements of ISO 15189, the standard report form for IFA-HEp-2 testing results was developed.Conclusions. To achieve high quality of interaction between clinical diagnostic/immunological laboratories and clinical departments of health care facilities, it’s necessary to introduce into practice a standard form for IFA-HEp-2 testing results, developed relying on modern clinical recommendations and interlaboratory consensus.

Rheumatoid arthritis (RA) is an autoimmune rheumatic disease of unknown etiology, characterized by chronic erosive arthritis (synovitis) and that can involve other tissues and organs. The use of biologics in clinical practice, including humanized monoclonal antibodies (IgG1) to interieukin-6 (IL-6R) receptors, it's the cause to search for predictors of response to this therapy. The aim of this study was to determine the relevance of multiplex cytokine analysis in evaluating the effectiveness of tocilizumab (TCZ) in RA. Materials and methods . We examined serum samples from 43 patients with RA. The comparison group was 297 healthy subjects matched by gender and age. Serum concentration of IgM and IgA rheumatoid factors (RF) and C-reactive protein (CRP) in serum was measured by immunonephelometry; of antibodies to cyclic citrullinated peptide (anti-CCP), antibodies to modified citrullinatedvimentin (anti-MCV), matrix metalloprotinase-3 (MMP-3) - by enzyme-linked immunosorbent assay; of cytokines - by xMAP technology. Results. At week 4 on TCZ therapy, patients with RA had a decrease serum levels of CRP, IgM RF, AMCV, MMP-3; on 8th - ESR, CRP, IgM and IgA RF, anti-CCP and anti-MCV, on the 24th - of all biomarkers, excluding anti-CCP. At 4th, 8th, 24th weeks of therapy, there was a decrease in serum levels of all studied cytokines (excluding IL-6 at 4th week and IL- 1ra at 8th and 24th weeks). The most associated factors witch effectiveness of TCZ are: MMP-3 (AUC0.7), anti-CCP (0.7) and VEGF (0.7). A predictive model based on the assessment of serum levels of this biomarkers can predicting the clinical response to TCZ in RA (AUC = 0.85; CI: 0.7-1.0). Conclusion. The data about association of basal serum concentrations of anti-CCP, MMP-3 and VEGF with the response in RA patients, made it possible to create a multiparameter index for predicting the TCZ effectiveness.

Inflammatory bowel disease IBD (Crohns disease CD, ulcerative colitis UC) immune-mediated diseases of the digestive tract of unknown etiology. The basis of the pathogenesis of IBD is a violation of the protective mechanisms of the intestinal barrier as a result of a complex interaction of environmental factors, a genetic predisposition and defects in the activation of the immune response in the lymphoid tissue of the intestinal mucosa. Three groups of antibodies are detected in the sera of IBD patients: autoantibodies, antimicrobial antibodies and antibodies to peptide antigens. In CD, the most useful diagnostic markers are ASCA; in UC patients pANCA. Antibodies are not among the diagnostic criteria for CD and UC, the diagnosis of which is traditionally made on the basis of a complex of clinical, radiological, endoscopic and histological signs, but can be used as useful additional non-invasive markers for early diagnosis, assessment of clinical phenotypes, prognosis and effectiveness of treatment of these diseases.

The detection in serum of monospecifc antibodies that induce a dense fne-speckled fluorescence when interacting with the DFS70 / LEDGF / p75 nuclear antigen is negatively associated with the development of systemic autoimmune rheumatic diseases (SARD) and increases the diagnostic specifcity of the screening study of antinuclear antibodies (ANA) using indirect immunofluorescence on HEp-2 cells (IIF-HEp-2). The results of assessing the clinical signifcance of anti-DFS70 antibodies vary depending on the test systems and the selection of patient groups. The aim of this work is to study the frequency of detection of monospecifc anti-DFS70 antibodies in blood serum in healthy individuals and patients with SARD. Sera of 74 healthy donors and 59 patients with SARD were studied (27 – systemic lupus erythematosus – SLE, 15 – Sjogren's syndrome – SjS, 17 – rheumatoid arthritis – RA). Classical antinuclear antibodies (ANA) and anti-DFS70 antibodies were determined by IIF using a mixture of standard and genetically engineered DFS70-KO HEp-2 cells that do not express DFS70 / LEDGF / p75 as a substrate. 14.9% of healthy donors and 83.1% of SARD patients (96.3% – SLE, 100.0% – SS, 47.1% – RA) were seropositive for antinuclear factor (ANF). Classical ANA with homogeneous, speckled, nucleolar, cytoplasmic, mixed types of fluorescence and the absence of anti-DFS70 antibodies were found in all ANF-positive patients with SARD and in 8.1% of healthy donors. Monospecifc anti-DFS70 antibodies without classical ANA were detected in 6.8% of healthy individuals and were absent in SARS. Among ANF-positive healthy donors, the frequency of isolated detection of anti-DFS70 antibodies was 45.5%. The detection of monospecifc anti-DFS70 antibodies can be considered as a potential predictive marker for excluding the diagnosis of SARD in ANF-positive patients with no or unclear clinical signs of these diseases.

Background: An important characteristic of immune pathology in rheumatoid arthritis (RA) is a B-cell tolerance defect, associated with autoantibodies production, and antigen-specific activation of Th-1 CD4+ T lymphocytes with an excess production of pro-inflammatory cytokines compared to anti-inflammatory ones. Pro-inflammatory cytokines contribute to the development of local inflammatory effects, induce bone destruction and pannus formation, and contribute to the development of autoimmune abnormalities and systemic manifestations. Anti-inflammatory cytokines are able to reduce the rate of joint destruction. There is evidence of the involvement of Th2 cytokines in the development of early RA. These facts suggest the need for a thorough investigation into the balance between the Th1 and Th2 types of immune response at different stages of the disease.Aim: To assess the importance of сytokine profiling in the evaluation of immune abnormalities in RA.Materials and methods: In this descriptive, controlled, retrospective study, we examined 118 patients with RA and 33 healthy donors as a control group. Serum IgM rheumatoid factor (RF) and C-reactive protein (CRP) levels were measured by immunonephelometry; anti-cyclic citrullinated peptide antibodies (anti-CCP) and anti-mutated citrullinated vimentin antibodies (anti-MCV) were determined by an enzyme immunoassay, cytokines levels with "xMAP" technique.Results: Serum cytokine levels vary depending on RA duration. The cytokine profile in early RA, unlike that in established RA with a duration of more than 6 months, is characterized by higher levels of pro-inflammatory (MIP-1α), Th1 (IFN-γ), and Th17 (IL-17) cytokines, colony-stimulating factors (IL-7, G-CSF), and chemokines (IL-8, IP-10) (p < 0.05 for all parameters). In established RA, the levels of pro-inflammatory (IL-1β, -6, -15, TNF-α), anti-inflammatory (IL-1ra, IL-10, IL-13, IL-5), Th1 (IL-2, IL-12), Th2 (IL-9) cytokines and colony-stimulating factors (G-CSF, GM-CSF) correlate with the concentrations of IgM RF and antibodies to citrullinated proteins (antiCCP, anti-MCV) (all p < 0.05). There was also а correlation between CRP and pro-inflammatory (IL-1β, IL-6, TNF-α), Th1 (IL-12), Th2 (IL-5, IL-9) cytokine levels and between DAS28 and pro-inflammatory cytokine (IL-6) and colony-stimulating factor (G-CSF) levels (all p < 0.05). Conclusion: In RA, cytokines, chemokines and colony-stimulating factors mirror the inflammatory activity of the disease. Changes in blood concentrations of cytokines enable to get an insight into the complex interplay of numerous mediators of innate and acquired immunity

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by pathological activation of the innate and acquired immune response, the formation of antinuclear antibodies (ANA), and dysregulation of cytokine production. Objective: to study the relationship of ANA and cytokine profiles in patients with SLE using multiplex immune analysis (MIA) of these biomarkers. We examined 94 patients with SLE (SLICC diagnosis criteria, 2012) and 28 healthy donors. Profiles of ANA and cytokines in blood serum were determined on the basis of suspension microarray technology xMAP. In SLE, antibodies to dsDNA (52.1 %), nucleosomes (54.3 %) and SS-A/Ro (37.2 %), less often to Sm (28.7 %), RibP (14, 9 %), RNP-70 (13.8 %) and SS-B/La (11.7 %). Disease activity (SLEDAI-2K) positively correlated with the concentration of antibodies to dsDNA (r = 0.6), nucleosomes (r = 0.7), Sm (r = 0.4) and RibP (r = 0.3) (p < 0.05). In the sera of patients with SLE, an increase in the levels of IL-4, -6, -8, -12, GM-CSF, MCP-1, MIP-1β, RANTES and a decrease in the content of IL-1β, IL-1ra, IL-2, IL-9, IL-10, eotaxin, G-CSF, IFN-γ, MIP-1α, TNF-α, FGF, PDGF-BB, VEGF compared to donors (p < 0.05). An increase in the concentration of IP-10 and MCP-1 was associated with high disease activity (r = 0.4; r = 0.3; p < 0.05), hyperproduction of antibodies to dsDNA (r = 0.3), nucleosomes (r = 0.5), Sm (r = 0.5), SS-B/La (r = 0.3), RibP (r = 0.4) (p < 0.05) and antibodies to Sm (r = 0.3), SS-B/La (r = 0.3), RibP (r = 0.3) (p < 0.05), respectively.Conclusion: the formation of ANA and high activity of SLE are associated with the overexpression of chemokines IP-10 and MCP-1 induced by IFN.

Aim: To assess serum levels of vascular endothelial growth factor (VEGF) and its clinical correlates in patients with systemic sclerosis (SSc). Materials and methods: Forty six (46) patients with SSc aged from 19 to 77 years (median, 50 years), with duration of the disease from 0.5 to 24 years (median, 7 years) were recruited into the study. There were equal numbers of the patients with limited (LSSc) and diffuse (DSSc) types of the disease (23 patients in each group, or 50%). All patients underwent clinical examination, including measurement of the forced vital capacity (FVC), diffusing capacity of the lung for carbon monoxide (DLCO) and pulmonary artery systolic pressure (PASP). Serum VEGF-A levels were determined by immunoenzyme assay in the patients and in 20 healthy controls. Results: VEGF levels in the healthy individuals were in the range from 0.2 to 264 pg/mL (median, 90.2). In SSc patients they varied from 0.02 to 1034.2 pg/mL (median, 147.2), with mean VEGF levels being over 2-fold higher than that in the control group (212.35 ± 253.93 and 97.74 ± 71.46 pg/mL, respectively; p = 0.032). DSSc patients had VEGF levels of 0.02 to 599.8 pg/mL (median, 93.6), whereas in LSSc they were from 0.02 to 1034.2 pg/mL (median, 162.4). Mean VEGF level in LSSc was higher than in DSSc (267.11 ± 268.74 vs 120.4 ± 141.09 pg/mL, respectively; p = 0.012). Current or past digital ulcers were found in 19 (41%) of all patients. Mean VEGF level in the patients with digital ulcers was higher than in those without ulcers; however, the difference was not statistically significant. PASP exceeded 30 mm Hg in 19 (43%) of the patients. VEGF levels in the patients with PASP of less than 30 mm Hg and ≥ 31 mm Hg were in the range of 0.02 to 363.6 pg/mL (median, 79.6) and 0.2–1034.2 pg/mL (median, 222.3), respectively. Mean VEGF level in the patients with high PASP was significantly higher than that in the patients with normal PASP (р = 0.0042). In the patients with DLCO ≥ 50% and < 50% serum VEGF levels were found to be 0.02 to 599.8 pg/mL (median, 59.75) and 0.02 to 1034.2 pg/mL (median, 195.9), respectively. Mean VEGF levels in the patients with DLCO of less than 50% was significantly higher than in the patients with DLCO of 50% and above (364.2 ± 381.95 and 128.55 ± 142.7, respectively, р = 0.034). FVC was decreased (< 80% of predicted) in 11 (26%) of 43 patients. Mean VEGF levels in the patients with low FVC was higher than in those with normal FVC, although the difference was non-significant. There was a moderate direct association between VEGF levels and PASP values (R = 0.4; p = 0.007). Also, a trend towards an inverse correlation between DLCO and VEGF levels was observed, which was however non-significant (R = -0.28; р = 0.07). Conclusion: A significant proportion of SSc patients have high serum VEGF levels. A close association with some clinical correlates indicates a pathogenetic role of VEGF in SSc. Further studies are necessary to clarify the precise contribution of VEGF into SSc pathophysiology.

Background Cytokines play an important role in the pathogenesis of SLE. TNF-a, IL-6 are the cytokines with suggested proinflammatory and immunoregulatory actions in the pathogenesis of SLE with differential effects on B or T cells, as well as on programmed cell death. Fas/APO-1 and Fas ligand (FasL) are involved in the apoptosis. FasL is a marker of apoptosis, it induces cell apoptosis when binds to Fas/APO-1. Both are expressed in membrane-associated and soluble forms (sFas/APO-1 and sFasL). sFas/APO-1 is known to inhibit the apoptosis via blocking the binding of Fas/APO-1 to FasL/sFasL. Objectives We evaluated the levels of IL-6 and TNF-α and their possible association with disease activity, apoptosis markers. Methods The study included 52pts (89% female, age 30,0 [26,5–44,5 years] (median [interquartile range 25% >75%])) with SLE ACR, 1997 and 20 controls (100% female without any rheumatic and infectious diseases, age 30,0 [25,0–39,5 years]). SLE-related factors, including disease duration, clinical features, SLE Disease Activity Index (SLEDAI 2K) and Systemic Lupus International Collaborating Clinics (SLICC) damage index were evaluated in parallel with relevant laboratory findings, autoantibodies. Serum levels of IL-6 and TNF-α (pg/ml), sFas/APO-1 (pg/ml) and sFasL (ng/ml) were measured by ELISA (Bender MedSystem GmbH, Austria). Results Median SLE duration was 5 [1–11] years, SLEDAI 2K–9,5 [4,0–16,0], SLICC-0 [0–1], current prednisone dose–5,0 [0–11,9] mg/day, 44% of pts received hydroxychloroquine, 17% >cyclophosphamide, 10% >azathioprine, 4% >mycophenolate mofetil, 8% >biologics. SLEpts had higher level of IL-6 vs control(2,9 [1,5–9,7] vs 1,4 [0,7–2,1] pg/ml, p<0,01) and lower level of TNF-α(1,9 [0,5–3,2] vs 4,5 [3,5–6,4] pg/ml, p<0,0001), lower levels of sFas/APO-1 vs control (657,5 [483,2–791,4] vs 1456,9 [426,5–2060,8] pg/ml, p>0,05) and lower levels of sFasL vs control (0,01 [0,01–0,03] vs 0,07 [0,05–0,10] ng/ml, p<0,0001). In SLE pts serum level IL-6 correlated with IgM (r=0,69, p<0,01), K+(r=0,43, p<0,01), TNF-α(r=0,50, p<0,001), APRIL(r=0,39, p<0,01) and sFas/APO-1 levels(r=0,29,p<0,05); TNF-α level correlated with SLICC(r=0,32, p<0,05), APRIL (r=0,42, p<0,01), IL-6(r=0,50, p<0,001), haemoglobin concentration(r=0,39,p<0,01), sFasL (r=0,44, p<0,01) and total cyclophosphamide dose (r=0,28,p<0,05). We divided SLEpts on two groups: the 1st-pts (n=31) with high activity (SLEDAI 2K≥8), the 2nd–pts (n=21) with low (SLEDAI 2K<8). The pts in the 1st group had lower level of TNF-α compared to 2nd(1,6 [0,3–2,2] vs 2,8 [1,3–4,5] pg/ml, p<0,05), with no difference in IL-6 level. Conclusions SLEpts demonstrated higher IL-6 and lower TNF-α levels as compared to healthy controls. Pts with high activity of SLE had decreased level of TNF-α. There is no correlation between disease activity and IL-6 level, as well as between elevated IL-6 and acute phase indicators. We suggest an immunoregulatory not proinflammatory role of IL-6 in SLE and probable protective role of TNF-α in SLE pathogenesis (it’s deficiency can lead to the development and high activity of SLE). There was no correlation between IL-6, TNF-α levels and any SLE features. The correlation of IL-6 with sFas/APO-1 level (apoptosis inhibitor that blocks binding of sFas/APO-1 to sFasL) and TNF-α with sFasL concentration (a marker of apoptosis) suggests their different roles in the mechanisms of apoptosis. Disclosure of Interest None declared

Objectives To assess the efficacy of a rituximab and belimumab combination therapy in pts with active SLE and dynamics of CD19+ B-lymphocytes count in treated pts. Methods The study included 7 SLE pts (1M/6F) with high (SLEDAI2K≥10 – 4pts.) and moderate (SLEDAI2K<10- 2pts.) disease activity; out of them 1 patient had lupus nephritis, 1- vasculitis, and remaining 5 had predominantly mucocutaneous and articular manifestations of SLE. The dose of oral GCs at baseline did not exceed 20 mg/day, two pts were treated with prednisone 5 mg/day. The damage index at baseline was 0 in 3 pts,≤1 in 3pts, and ≥1 – in 1 patient. Rituximab (RTM) was administered at 500–1000 mg, with subsequent adding of Belimumab (BLM) 3 months later at a standard dosing regimen 10 mg/kg once a month during 9mo. CD19+ B- lymphocytes counts were obtained before initiation RTM (0), and subsequently after 3, 6, 9, and 12mo. Depletion of CD19+ B- lymphocytes after RTM was assessed as the decrease of B-cell counts after 3mo<0,01 10*9/l, where 0 10*9/l was categorised as complete depletion, from 0001 to 0,01 10*9/l – partial depletion, and >0,011 10*9/l – absence of depletion. The comparison group included 20 pts receiving a single 500–2000 mg dose of RTM. Results 6 pts demonstrated the decrease in clinical and laboratory SLE activity, starting from 3mo of follow-up (SLEDAI-2K 0 mo–Me 10, 9;16 3mo-Me 8, 4;86mo–Me 4, 2;6 9mo–Me 5,4;10 12mo–Me 2 2;6) with RTM+BLM combination therapy. The oral GCs dose was reduced to 7,5 5;10mg/day by 12mo, none of the patient required prednisone dose escalation during follow-up. There were no cases of severe infection. The damage index did not increase by 12mo. The combination therapy reduced the absolute counts of CD19+. B-cells. RTM therapy resulted in complete depletion in 2 pts, in partial depletion – in 3; in 1 patient the depletion was not documented. Addition of BLM resulted in slowing down of CD19+ B-cell repopulation (figure 1) among pts with complete and partial depletion (0mo–Me 0,11 × 109/l[0,1;0,28], 12mo -Me 0,01 × 109/l[0,0085; 0025]) vs pts receiving RTM monotherapy (0mo–Me 0,1 × 109/l[0,06;0,2], 12mo -Me 0,03 × 109/l[0,008; 0,08]). The decrease in CD19+ B-cell counts after RTM was also documented in the patient who didn’t develop depletion initially (0mo–0,5 10*9/l, 12mo-0,04 10*9/l). RTM and BLM combination failure, as well as failure of standard GCS and cytostatic based therapy, was documented in one patient with cutaneous, articular and haematological SLE. Conclusions Combination therapy allows to gain control over disease activity in short time, due to the effect of RTM, while added BLM provides further prolongation of the effect achieved, minimising the risk of exacerbation. This combination may be used as a method of choice in pts with severe SLE involving vital organs, and in persistent cutaneous-articular disease and high immunological activity. Of notice is the fact, that use of RTM and BLM combination allowed to manage all pts on low and medium doses of GCs starting from RTM and BLM initiation and during the entire follow-up, thereby reducing the risk of irreversible organ damage. In these patients there were no signs of infection. Disclosure of Interest None declared

Background Monotherapy with MTX is the first step of RA therapy as recommended by EULAR. According to “Treat to Target” (T2T) strategy principles insufficient response to MTX requires prompt switch to a combination therapy with biological agents. Objectives To find out whether polymorphisms of immune response genes are associated with early prescribing the biological treatment in eRA pts, refractory to the SC MTX monotherapy. Methods By January 2014, 210 pts with RA were included in the REMARCA study (Russian InvEstigation of MethotrexAte and biologics in eaRly aCtive inflammatory Arthritis), and 88 pts have passed the 12 months control point. All pts started SC MTX monotherapy with rapid up-titration of the dose from 10 to 25–30 mg/week. Therapy was revised every 3 months using DAS28, SDAI and CDAI indices. Combination with biologics (in most cases TNF inhibitors) was used in 57 (65%) of pts at (median) 3 [3;6] month [1]. Retrospectively, 45 out of 88 active eRA pts (35 woman, mean age 53,5; 46–59,5 years, mean disease duration 7,0; 4,0–11,5 months, mean DAS28 5,8; 4,9–6,4, mean hs-CRP 27,0; 9,7–61,0 mg/l) were selected to evaluate the changes in cytokine profile in MTX-naive pts during SC MTX and SC MTX + adalimumab (SC MTX+ADA) therapy [2]. These 45 pts were genotyped for the following gene polymorphisms (SNPs): PTPN22 (+1858 C/T, rs2476601), CTLA4 (+49A/G, rs231775), TNFAIP3 (rs675520, rs6920220, rs10499194), IL6 (-174G/C, rs1800795), IL6R (+358A/C, rs8192284), TNFA (-308A/G, rs1800629), MCP1/ CCL2 (+2581A/G, rs1024611), IL10 (-592A/C, rs1800872, -1082 A/G, rs1800896), IL1A (-889C/T, rs1800587), IL1B (+3953C/T, rs1143634). Results By the end of 3 months of SC MTX therapy, 23 out of 45 eRA pts (51,1%) adequately responded to SC MTX (EULAR criteria) and continued on SC MTX monotherapy. In 22 pts (48,9%) SC MTX monotherapy failed, thus they were switched to SC MTX + ADA combination therapy. CTLA-4 gene polymorphism (+49A/G) was the only predictor for the administration of biological therapy in eRA pts, that was confirmed by a logistic regression analysis [OR=7,7 95% CI 1,4–40,9, p=0,017]. The carriers of at least one G allele (AG/GG genotypes) received the biological therapy more often than subjects with AA genotype (20/22, 90,9% and 13/23, 56,5% respectively). Moreover, the CTLA-4 (+49A/G) genotypes (AG/GG vs AA respectively) were associated with DAS28 (3,6±1,1 and 4,6±1,6, p=0,04), the number of tender joints (3,1±3,1 and 7,1±5,1, p=0,016), the number of swollen joints (2,7±2,5 and 5,3±4,9 p=0,024), SDAI (11,1±7,2 and 20,2±12,1, p=0,018), CDAI (10,6±7,1 and 18,6±10,6, p=0,025) and CRP (6,3±12,1 and 21,8±37,1 p=0,018) values after 3 months of SC MTX monotherapy. Conclusions Our data suggest that CTLA-4 (+49A/G) genetic polymorphism is associated with more severe rheumatoid arthritis and may predict the need for early administration of biological therapy. References Karateev D., E. Luchikhina E., N. Demidova N. et al. Ann Rheum Dis 2014;73(Suppl2): 235–236. Avdeeva A.S. Novikov A. A, Aleksandrova E. N. et al. Ann Rheum Dis 2014;73(Suppl2): 215–216. Disclosure of Interest None declared

Objective: to estimate changes in the cytokine profile in patients receiving abatacept (ABC). Subjects and methods. The investigation enrolled 44 patients with rheumatoid arthritis (RA) who had been unsuccessfully treated with disease-modifying antirheumatic drugs and biological agents. A control group included 16 healthy donors. The majority of patients were women who were positive for rheumatoid factor (RF) (80%) and antibodies to cyclic citrullinated peptide (ACCP) (79.5%); the mean age was 49.6±13.9 years; the median disease duration was 2 [1.4; 3] years with high RA activity (mean DAS28, 5.2±0.8). The serum concentrations of interleukin (IL) 1β, IL-6, IL-17AF, tumor necrosis factor-α (TNF-α), VEGF-A, IP-10, and YKL-40 were measured by enzyme immunoassay before and 6 months after ABC therapy. Disease activity was assessed using DAS28 every 3 months. ABC was infused intravenously according to the standard regimen. Results and discussion . The patients with RA as compared with the control group had significantly elevated levels of IL-6 (2.4 [1.1; 6.4] and 0.7 [0.62; 1.0] pg/ml; p=0.0002), YKL-40 (97 [68.4; 97.9] and 64 [52.4; 107.5] pg/ml; p=0.03), IP-10 (21 [12.9; 49.8] and 14 [9.2; 15.2] pg/ml, respectively; p=0.005). ABC caused a significant decrease in RA activity after 3 months of therapy (p<0.05). Following 6 months, 86% of the patients achieved good and moderate EULAR responses; low RA activity according to DAS28 was recorded in 52%. ABC induced significant decreases in the concentrations of IL-6 to 1.29 [0.9; 2.2] pg/ml (p=0.0006) and IP-10 to 14 [7.5; 28] pg/ml (p=0.007) after 6 months of therapy. A similar trend was observed when assessing changes in the concentration of matrix metalloproteinase 3 (MMP-3), which reduced from 30.1 [13; 82] to 10 [7.4; 55] pg/ml (p=0.0003), and in that of RF, which declined from 218 [9.6; 187] to 159 [9.7; 155] pg/ml (p=0.02). The lower levels of IL-6 (r=0.5) and IP-10 (r=0.32) significantly correlated with a decrease in DAS28 (p<0.05). There was a trend towards a more pronounced reduction in disease activity in patients positive for ACCP and antibodies to modified citrullinated vimentine (AMCV). The percentage of non-responders to therapy in the ACCP- and AMCV-negative groups was nearly twice as high as in those who were positive for these antibodies (27.2 and 16%; 26.7 and 14.8%, respectively), but these differences failed to reach significance. However, after 6-month of follow-up, the percentage of non-respondents in the AMCV-negative group was significantly higher than in the AMCV-positive group (20% and 0%, respectively; p=0.03). The patients who did not respond to ABC therapy had higher baseline levels of IL-6 (p=0.03) and YKL-40 (p=0.02). Conclusion. ABC therapy results in a substantial reduction in the concentration of the proinflammatory cytokines IL-6 and IP-10, as well as MMP-3 and RF. The lower levels of IL-6 and IP-10 significantly correlated with a decrease in RA activity. There was a tendency towards a more pronounced reduction of disease activity in ACCP- and AMCV-positive patients. The high baseline levels of IL-6 and YKL-40 and the absence of AMCV may suggest that ABC therapy can be ineffective.

Ankylosing spondylitis (AS) is a chronic inflammatory disease from a group of spondyloarthritis (SpA), which is characterized by lesions of the sacroiliac joints and spine with the common involvement of entheses and peripheral joints in the pathological process. Advances in modern laboratory medicine have contributed to a substantial expansion of the range of pathogenetic, diagnostic, and prognostic biomarkers of AS. As of now, there are key pathogenetic biomarkers of AS (therapeutic targets), which include tumor necrosis factor-α (TNF-α), interleukin 17 (IL-17), and IL-23. Among the laboratory diagnostic and prognostic biomarkers, HLA-B27 and C-reactive protein are of the greatest value in clinical practice; the former for the early diagnosis of the disease and the latter for the assessment of disease activity, the risk of radiographic progression and the efficiency of therapy. Anti-CD74 antibodies are a new biomarker that has high sensitivity and specificity values in diagnosing axial SpA at an early stage. A number of laboratory biomarkers, including calprotectin, matrix metalloproteinase-3 (MMP-3), vascular endothelial growth factor, Dickkopf-1 (Dkk-1), and C-terminal telopeptide of type II collagen (CTX II) do not well reflect disease activity, but may predict progressive structural changes in the spine and sacroiliac joints in AS. Blood calprotectin level monitoring allows the effective prediction of a response to therapy with TNF inhibitors and anti-IL-17А monoclonal antibodies. The prospects for the laboratory diagnosis of AS are associated with the clinical validation of candidate biomarkers during large-scale prospective cohort studies and with a search for new proteomic, transcriptomic and genomic markers, by using innovative molecular and cellular technologies.

Background Concurrent infections turn out to be the second leading cause of death in systemic lupus erythematosus (SLE) pts after SLE per se. Immunization of SLE pts with pneumococcal vaccine is an important prophylactic approach to prevent severe lower respiratory tract (LRT) infections in SLE pts. Objectives To study the relevance of 23-valent pneumococcal vaccine for immunization of SLE pts. Methods The study included 30 SLE pts, 27 females, 3 males, aged 19 - 62 y. Duration of follow up (FUP) was 12 months in 24 pts, and 7–10 months – in 6 pts. High disease activity at the time of immunization was documented in 1 patient, low activity – in 20 pts, moderate – in 4 pts, and remission – in 5. 29 pts were treated with glucocorticosteroids (GCs), 23 – with hydroxychloroquine, 14 pts – with cytostatic (CS) agents. Twelve pts were on biological disease-modifying antirheumatic drugs (bDMARDs). One dose (0,5 ml) of 23-valent polysaccharide pneumococcal vaccine was administered subcutaneously. The duration of FUP was 7–12 months. Control visits were scheduled as follows: at baseline (Visit 1), at 1st, 3rd, and 12th months (Visit 4) after immunization. Standard clinical examination and lab tests, including blood immunology, were performed at each visit. Vaccine immunogenicity was evaluated based on the level of serum antibodies (AT) to Streptococcus pneumoniae capsular polysaccharide (VaccZymeTM PCP Ig 2 panels (The Binding Site Ltd, Birmingham, UK)) – 4 times during 1 year. Results No post-immunization complications were seen in 11 (36,7%) pts, local reactions of varying intensity lasting from 2 to 7 days were documented in 18 (60%) pts. One patient (3,3%) developed the local type III hypersensitivity reaction known as Arthus phenomenon. All symptoms subsided within 7 days after administration of antihistaminic agents and local GCs. Not a single vaccination-related SLE exacerbation episode was documented in 24 pts during the FUP. Significant (≥2-fold vs baseline) increase of serum AT levels to S. pneumoniae polysaccharide was observed during the FUP (Table). In 10 (41,7%) out of 24 pts (“non-responders”) more than 2-fold increase of anti- S. pneumoniae ATs was not achieved by 12th month of FUP. Among them 7 (70%) pts were receiving bDMARDs. 4 (28,6%) out of 14 “responders ” were also treated with bDMARDs. Non-severe pneumonia was documented in 2 out of 24 pts within1 year after vaccination; both cases successfully resolved after 7- and 5-days of oral antibiotic treatment in an out-patient setting. Both pts had episodes of pneumonia in past medical history. One of them had SLE-induced interstitial lung disease. This patient was treated with GCs, mycophenolate mofetil, and rituximab, no post-vaccination response was documented. The second patient had two previous pneumonia episodes, she demonstrated 4–5-fold increase in anti- S. pneumoniae AT titre; her therapy included GCs (10 mg/day) and hydroxychloroquine. There were no clinical or radiological symptoms of pneumonia in remaining 22 pts during the whole FUP. Conclusions Obtained results are indicative of good tolerability, safety and immunogenicity of 23-valent pneumococcal vaccine in SLE pts. Further studies are necessary for more comprehensive evaluation of vaccine clinical efficacy. Disclosure of Interest None declared

Objectives To study B-cell subpopulation dynamics in SLE patients following Rituximab (RTX) therapy. Methods The study included 31 SLE pts (3m/28f) with high (SLEDAI2K≥10–28 pts.) and moderate (SLEDAI2K<10–3 pts.) disease activity; out of them 12 pts with SLE nephritis, 5 pts with neurolupus and 8 with vasculitis. RTX was administered to pts who failed to respond to glucocorticoids (GCs) and cytostatics (CTs). B-cells subpopulations were assessed before RTX administration (Mo0), and at Mo3 and Mo6 of RTX therapy. RTX was administered at 500 to 2000 mg doses depending on disease activity. The absolute counts of CD19+ B-cells, the total population of memory B-cells (CD19+CD27+), “preswitch” (CD19+IgD+CD27+) and “postswitch” (CD19+IgD-CD27+) memory B-cells, “naive” (CD19+IgD+CD27-), plasma cells (CD19+CD38+) and double negative B-cells (CD19+CD27-IgD) were measured. All B cell subsets were analyzed with multicolor flow cytometry using a panel of monoclonal antibodies to B-lymphocytes9 surface membrane markers. Results Following initiation of RTX SLE clinical and lab activity indices have decreased in all 31 pts by Mo3 and Mo6 of follow up (SLEDAI-2K Mo0–Me 15 [12;18], Mo3-Me 6 [4;10], Mo6–Me 4 [2;8]), as well as absolute count CD19+ B-cell population (Mo0–Me 0,119x109/l [0,05;0,26], Mo3–Me 0x109/l [0;0,003], Mo6-Me 0,004x109/l [0;0,02]). B-cell repopulation by Mo6 in 15 out of 31 pts without signs of relapse and 4 pts with earlier relapse SLE was dependent on “naïve” B-cells (Me 0,0015x109/l [0,0002;0,01] vs Me 0,006x109/l [0,0033;0,008]), double negative (Me 0,001x109/l [0,0002;0,002] vs Me 0,0035x109/l [0,0018;0,005]) “postswitch” (Me 0,0005x109/l [0,00008;0,003] vs Me 0,0012x109/l [0,0003;0,0035]) and “preswitch” memory B-cells (Me 0,0006x109/l [0,00007;0,001] vs Me 0,0023x109/l [0,0005;0,005]). Conclusions Decrease in clinical and lab SLE activity was documented in all 31 pts by Mo3 after one course of RTX therapy. In 4 pts with earlier relapse SLE at Mo6 B-cell was found repopulation and significant increase “naïve” B-cells, double negative, “postswitch” and “preswitch” memory B-cells compared with the group without relapse. Disclosure of Interest None declared

Aim. To examine the association of signal transducer and activator transcription 4 (STAT4) rs7574865 G/T polymorphism with a predisposition to systemic sclerosis (SSC) and associated clinical and autoimmune phenotypes in a Russian population. Subjects and methods. A total of 102 patients with SSC and 103 healthy individuals as controls were examined. STAT4 rs7574865 polymorphism was investigated by real-time polymerase chain reaction. Results. The carriers of the T allele showed a statistically significant association with SSC, a diffuse form (DF), the presence of interstitial lung disease (ILD), cardiac injury (CI), and seropositivity for anti-topoisomerase I antibodies (ATA). Conclusion. The findings results confirm the important role of STAT4 gene in the predisposition to SSC and its phenotypes, such as DF, ILD, CI, and ATA in the Russian population.

Quantitative evaluation and assessment of pharmacokinetic parameters of Diprospan® (suspension for injection 7mg/mL (2mg+5mg/mL) of betamethasone) were performed in urine samples taken from patients with rheumatoid arthritis or ankylosing spondylitis for 28days after systemic intramuscular administration in routine clinical practice in an open-comparative prospective cohort study. The maximum betamethasone concentration was reached at day 4 of the follow-up; in some cases, β-phase of elimination of the drug was appeared at day 14 or at day 21 of the follow-up. The deferred β-phase elimination was likely a consequence of the physiological characteristics of the patients or of the influence of non-steroidal agents. The half-life of betamethasone was 8.5days. The elimination rate constant was 2.49h-1; the mean clearance was 4.72L/d. The recommended frequency of the drug administration to its complete elimination was estimated up to 48days. Mann-Whitney test showed no significant differences in pharmacokinetic characteristics between male and female subjects. The prolonged elimination phase was observed in patients with deviations in their body mass index, continual treatment by diclofenac and nimesulide or, possibly, after consuming an alcohol. The study was recorded in Clinical Trials open source with identifier NCT03119454.

The FAS antigen (Apo-1/CD95) is a key molecule of apoptosis in most cell types, including activated immune cells and fibroblasts. The FAS gene promoter region contains a single-nucleotide polymorphism (-670A/G) associated with a substitution of the nucleotide arginine for guanine, which is associated with the predisposition to systemic lupus erythematosus, multiple sclerosis, sarcoidosis, and autoimmune hepatitis.Objective: to test the hypothesis that the FAS -670А/G polymorphism may predispose to systemic sclerosis (SS), its clinical and autoimmune phenotypes in a Russian patient sample.Subjects and methods. The instigation enrolled 90 SS patients who were classified according to clinical and autoimmune phenotypes. A control group consisted of 152 apparently healthy unrelated individuals matched for sex and age. The FAS -670А/G polymorphism was studied by polymerase chain reaction, followed by restriction fragment length polymorphism analysis.Results and discussion. A relatively small sample of Russian patients showed no statistically significant association of the studied FAS -670 A/G polymorphism with the predisposition to SS as a whole and the majority of its clinical and immunological phenotypes. There was a statistically significant positive association of the G allele (the FAS -670 GG+GA genotype) with the presence of digital ulcers and the chronic course of the disease. The G allele (the FAS -670 GG+ GA) was detected less frequently in patients with interstitial lung disease (ILD) than in those without ILD.Conclusion. The findings show that the FAS -670A>G polymorphism plays a role in the predisposition to some clinical phenotypes of SS in Russian patients.

Laboratory medicine in the early 21st century has achieved advances due to the development and prompt practical introduction of innovative molecular cell technologies, which have assisted in increasing the diagnostic sensitivity and specificity of laboratory tests and in substantially expanding the spectrum of study biomarkers in rheumatology. High-technology automated analytical systems using both classical uniplex methods for immunochemical analysis (indirect immunofluorescence test, enzyme immunoassay, immunoblotting, immunodot assay, immunonephelometry, chemiluminescence immunoassay, and radioimmunoassay) and multiplex diagnostic platforms based on DNA, RNA, protein and cellular microchips, polymerase chain reaction, flow cytometry, and mass spectrometry have been used in the past decade to determine biomarkers of rheumatic diseases (RD) in blood, synovial fluid, urine, biopsy specimens of the synovial membrane, kidney, and other affected tissues.Present-day generation of molecular and cellular biomarkers (autoantibodies, acute-phase inflammatory proteins, cytokines, chemokines, vascular endothelial activation markers, immunoglobulins, complement components, lymphocyte subpopulations, osseous and cartilaginous tissue metabolic products, intracellular signaling molecules, proteases, and genetic, epigenetic, and transcriptomic markers) is an important tool for prevention, early diagnosis, assessment of disease activity, progression rate, clinical laboratory subtypes of RD, prediction of the efficiency of therapy and the risk of adverse events during treatment. Deciphering of the key pathogenetic mechanisms of RD could identify the molecular and cellular biomarkers that might be used as therapeutic targets. Biologicals (monoclonal antibodies and hybrid protein molecules) that selectively inhibit proinflammatory cytokines and membrane molecules mediating the pathological activation of immunocompetent cells are successfully used to treat RD today.The alternative therapies of RD include the use of low-molecular-weight chemically synthesized agents that suppress the activity of tyrosine kinases. The important area of this therapy is to restore immunological tolerance and to correct autoimmune disorders by means of autologous hematopoietic stem cells, mesenchymal stromal cells, autologous tolerogenic dendritic cells, regulatory T and B cells, gene therapy, and peptide antigens. The prospects for the laboratory diagnosis of RD are associated with the necessity of harmonizing and standardizing the current methods to determine autoantibodies and with the search for and clinical validation of novel proteomic, transcriptomic, and genomic biomarkers.