[Fifteen-year results of bilateral and unilateral internal mammary artery grafting]. / Pyatnadtsatiletnie rezul'taty revaskulyarizatsii miokarda s ispol'zovaniem dvukh i odnoi vnutrennei grudnoi arterii.

OBJECTIVE: To evaluate the clinical effectiveness of bilateral internal mammary artery grafting over long-term (15 years) postoperative period. MATERIAL AND METHODS: There were 276 patients divided into two groups: 135 patients (group A) underwent bilateral internal mammary artery grafting and 141 patients (group B) underwent unilateral internal mammary artery grafting together with venous bypass grafts. On-pump surgeries and cardioplegia, parallel CPB and on-pump procedures were performed in equal proportions. Mean age of patients was 57.3±7.6 years. Diabetes mellitus was detected in 21 (15.5%) and 24 (19.1%) patients, respectively (p>0.05). Mean LV ejection fraction was 55.4±9.9%, revascularization index - 3.1±0.8 and 3.0±0.7, respectively. In the 1st group, 43 patients underwent bilateral internal mammary artery grafting alone. Autovenous grafts were additionally used in other 84 patients. RESULTS: Ten-year survival exceeded 90% in both groups. Freedom from adverse cardiac events after 15 years was significantly higher in group A (77.3% vs. 59.3%, p=0.018). In group A, 16 patients died throughout this period due to cancer (50%), myocardial infarction (12.5%), stroke (18.8%) and complications of diabetes mellitus (6.3%). In group B, 22 patients died mainly from cardiac causes (myocardial infarction - 40.9%, cancer - 27.3%). CONCLUSION: Bilateral internal mammary artery grafting has obvious advantages over traditional coronary artery bypass grafting. If we take into account higher proportion of cardiac causes in structure of mortality in group B, we can talk about positive impact of bilateral internal mammary artery grafting not only on the quality of life, but also on life expectancy in long-term postoperative period.

Diagnostic value of screening methods for the determination of antinuclear antibodies using indirect immunofluorescence on HEp-2 cells and enzyme immunoassay in autoimmune liver diseases.

Antinuclear antibodies (ANA) are a heterogeneous group of autoantibodies that react with various components of the cell nucleus and cytoplasm. ANA is the main serological marker for autoimmune liver disease (AILD). The aim of the study was to compare the diagnostic value of two methods of screening for the determination of ANA (indirect immunofluorescence reaction on HEp-2 cells (IIF -HEp-2) and enzyme-linked immunosorbent assay (ELISA) in the sera of AILD patients. The sera of 118 patients with AILD (51 with autoimmune hepatitis - AIH, 19 with primary biliary cholangitis - PBC, 48 with overlapping syndrome - OVERLAP), 30 patients with non-alcoholic fatty liver disease (NAFLD) and 30 healthy donors (HD) were studied. Determination of ANA by the IIF-HEp-2 method was carried out by visual assessment of samples under an AXIOSKOP 40 microscope, by ELISA - on an Alegria automatic analyzer. A weak degree of agreement between the positive and negative results of the ANA screening study using IIF-HEp-2 and ELISA (Cohen's kappa coefficient æ=0.4) was noted. Screening determination of ANA in patients with AILD by the IIF-HEp-2 method was distinguished by greater diagnostic sensitivity (DS) (68.6%) and a lower frequency of false negative results (31.4%) compared with ELISA (35.6% and 64.4 % respectively, p<0.05). The overall diagnostic specificity (DS) of the ANA study in IIF-HEp-2 was lower than with ELISA (66.7% and 86.7%, respectively, p<0.05). Both screening methods for determining ANA (IIF-HEp-2 and ELISA) were useful for diagnosing AILD (positive likelihood ratio - LR+: 2.1 and 2.6, respectively). In terms of the negative likelihood ratio (LR-), screening for ANA by the IIF-HEp-2 method, in contrast to ELISA, served as a "useful" test to exclude the diagnosis of AILD (0.5 and 0.8, respectively). The determination of ANA using IIF-HEp-2 is the most sensitive and "useful" screening test for the diagnosis of AILD, and ELISA is classified as a less "useful" screening method due to low diagnostic sensitivity and a high false-negative rate.

Adaptation of Microarray Assay for Serum Amyloid a Analysis in Human Serum.

Serum amyloid A is an inflammatory biomarker whose concentration changes during infectious and inflammatory diseases. SAA's tendency for aggregation and complex formation makes it difficult to determine its concentration in samples, especially when there is an increased level of it. Immunofluorescence SAA determination on a microarray was adapted for SAA quantification in human serum. Both the procedure and the diluent for the calibrator samples were chosen to obtain a dynamic range between 1 and 100 µg/mL. Mixtures of animal (rabbit, goat, mouse) sera with recombinant antigen diluted in certain concentrations were used for the calibrator samples. The method was tested using serum samples from 15 patients with rheumatoid arthritis or ankylosing spondylitis and 9 healthy donors. The results obtained on the microarray demonstrated a good correlation with the results determined by ELISA (Pearson's correlation coefficient is 0.93). The method developed could be a convenient tool for assessing SAA levels in a number of diseases, such as rheumatoid arthritis or infections of various etiologies, characterized by a significant increase in the level of this protein in the blood. The use of a microarray for the analysis allows the determination of the SAA concentration simultaneously with other inflammatory biomarkers.

[Adaptation of Microarray Assay for Serum Amyloid A Analysis in Human Serum].

Serum amyloid A is an inflammatory biomarker whose concentration changes during infectious and inflammatory diseases. SAA's tendency for aggregation and complex formation makes it difficult to determine its concentration in samples, especially when there is an increased level of it. Immunofluorescence SAA determination on a microarray was adapted for SAA quantification in human serum. Both the procedure and the diluent for the calibrator samples were chosen to obtain a dynamic range between 1 and 100 µg/mL. Mixtures of animal (rabbit, goat, mouse) sera with recombinant antigen diluted in certain concentrations were used for the calibrator samples. The method was tested using serum samples from 15 patients with rheumatoid arthritis or ankylosing spondylitis and 9 healthy donors. The results obtained on the microarray demonstrated a good correlation with the results determined by ELISA (Pearson's correlation coefficient is 0.93). The method developed could be a convenient tool for assessing SAA levels in a number of diseases, such as rheumatoid arthritis or infections of various etiologies, characterized by a significant increase in the level of this protein in the blood. The use of a microarray for the analysis allows the determination of the SAA concentration simultaneously with other inflammatory biomarkers.

Analysis of rheumatoid factor and acute phase proteins using microarrays in patients with rheumatoid arthritis.

One of the biomarkers of biggest clinical importance in rheumatoid arthritis (RA) is rheumatoid factor (IgM RF). The rheumatoid factor has insufficient sensitivity and specificity, therefore, to increase the diagnostic information of the test, acute phase proteins were used as concomitant biomarkers. Using biological microchips, we measured IgM RF, C-reactive protein (CRP) and Serum amyloid protein A (SAA) in patients with RA (n = 60), ankylosing spondylitis (AS) (n=55), systemic lupus erythematosus (SLE) (n=20) and healthy donors (HD) (n=9). It was shown that the medians of IgM RF concentrations are significantly higher (p<0.01) in patients with RA compared to patients suffering from other diseases and healthy donors. CRP and SAA were also significantly increased (p<0.05) in patients with RA and AS compared with SLE and HD. It has been shown that the complex determination of three biomarkers in differentiating RA patients with the comparison group had a higher diagnostic sensitivity than the isolated determination of IgM RF, while the addition of SAA makes the greatest contribution to improving the diagnostic characteristics of the biomarker panel: the use of a logistic regression model based on IgM RF and SAA allowed to increase the diagnostic sensitivity of the analysis from 58.3% to 65%. Thus, the developed microarray-based method can be used to detect and elucidate the diagnostic characteristics of RA biomarkers; however, further use requires validation of the obtained results on an expanded sampling.

[Clinical value of antibodies in inflammatory bowel diseases].

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.

Diagnosis of IgG4 - related ophthalmic disease in a group of patients with various lesions of the eye and orbits.

AIM: To provide demographic, clinical, laboratory, ultrasound, radiological, morphological/ immunomorphological phenotype of IgG4-related ophthalmic diseases, which allowsmaking a differential diagnosis with granulomatous, autoimmune, inflammatory, endocrine and hematologic diseases affecting the eye and orbits. MATERIALS AND METHODS: From 2004 to 2016 108 (78.2%) of the 138 patients were diagnosed with non-tumoral lesions of eye and orbits. In 48 patients (35%) at admission and 5 patients in the follow were diagnosed IgG4-related ophthalmic disease. In the analysis of 82 (f-44, m-38) patients with IgG4-related disease, localization of lesions in orbit observed in 53 (f-36, m-17) and it was the most frequent involvement in patients with IgG4-related disease (64.5%). Only 7 patients had isolated IgG4-related ophthalmic disease, whereas 46 patients (87%) had involvement of 2-7 locations, as a manifestation of IgG4-related systemic disease.During the examination, the average age of patients with IgG4-related ophthalmic disease was 47.5 years (19-73 years). Median time to diagnosis was 52.8 months before 2004 and 36 months 2004-2016. RESULTS: We noted the predominance of females in the ratio 2: 1 inthe group of patients with IgG4-related ophthalmic disease. Edema of the eyelids, nasal congestion (55-60%), tumor-like formations of the upper eyelids and increased lacrimation prevailed at the onset of the disease, whereas such functional impairment like limited mobility and pain in eyeballs, exophthalmos, ptosis and diplopia appeared later at 15-38% with a loss visual acuity in one case. Bilateral lesion (86%), mainly affecting the lacrimal glands (93.5%), infiltration of the ex- traocular muscles (83.5%) and retrobulbar tissue with a thickening of the optic nerve in one third of patients were the main localizations IgG4-related ophthalmic disease. Clinical symptoms were accompanied by the appearance of moderate inflammatory activity (38%), in- creased levels IgG (44%), IgG4(88%) and IgE (61%). Indicators of autoimmune disorders observed in 6-22% of patients, most often in pa- tients with simultaneous involvement of the salivary glands. Significant lymphoplasmacytic infiltration (94%) with a ratio of plasma cells (IgG4/IgG) secreting IgG4> 40% (90%) with fibrosis formation (94%) and follicle formation (71%) with a moderate amount of eosinophils (34%) were the major morphological / immunomorphological manifestations of IgG4-related ophthalmic disease. Signs of vasculitis and obliterative phlebitis were found in a small amount of patients. CONCLUSION: Determination of elevated levels of IgG-4 / IgE in patients with edema, pseudotumor of the eyelid, sinusitis and increase of the palpebral lobe of the lacrimal gland suggests the presence of IgG4-related ophthalmic disease. Minimally invasive incisional biopsy of lacrimal glands and salivary glands followed by morphological / immunomorphological research is needed for the correct diagnosis. Diagnostic orbitotomy in ophthalmic hospitals in such cases is inexpedient, since it leads to the development of dry eye. Massive lymphoplasmacytic infiltration with IgG4 / IgG ratio more than 40%, advanced fibrosis in biopsiesof the orbits tissue or salivary glands when combined lesions are required for the making the diagnosis of IgG4-related ophthalmic disease.

[Modern look at the problems of investigation of antinuclear antibodies in systemic lupus erythematosus (literature review).]

In the review, topical aspects of the study of antinuclear antibodies (ANA) in systemic lupus erythematosus (SLE) are considered. ANA is the main serological marker of SLE. In the sera of patients with SLE, antibodies to DNA, histones, nucleosomes, extractable nuclear antigens (Sm, U1 ribonucleoprotein, Ro / SSA, La / SSB, ribosomal protein P), nucleolar antigens and other cellular structures are detected. The ANA study using indirect immunofluorescence on HEp-2 cells (IIF-HEp-2) is recommended as a standard screening test for the diagnosis of SLE. The use of automated systems for the interpretation of cellular fluorescent tests contributes to the standardization and improvement of the reproducibility of the IIF. A new international nomenclature of types of nuclear, nucleolar (nucleolar), cytoplasmic and mitotic luminescence of ANA in IIF-HEp-2, including 28 variants of anticell ("Anti-cell" - AC) patterns was developed. In the practice of clinical diagnostic laboratories, high-performance automated methods for the determination of ANA based on ELISA, immunoblot, fluorescent, chemiluminescent and multiplex immunoassay are widely used. New mono- and multiplex methods of solid-phase analysis are expediently used as confirmatory reflex tests for the detection of varieties of antigen-specific ANA in patients with SLE with positive results of IIF-HEp-2. Identification of ANA profiles using multiplex technologies is a useful tool for implementing a personalized approach to diagnosis, evaluation of activity, prognosis, clinical and immunological subtypes, and the effectiveness of SLE therapy. The need for an ANA study not only to confirm the diagnosis of SLE, but also to identify the disease in the early and preclinical stages with the intention to prevent the development of the pathological process is discussed. Detection of monospecific anti-DFS70 antibodies allows to exclude the diagnosis of SLE inANA IIF-HEp-2 positive subjects. Presented is a modern algorithm for testing ANA with SLE.

[Clinical value of multiplex immune assay of antinuclear antibodies in systemic lupus erythematosus.]

A promising trend in the diagnosis of systemic autoimmune diseases is the multiplex immune assay (MIA) of autoantibodies and other laboratory biomarkers using microchips. The aim of the work was to study the diagnostic and prognostic significance of MIA antinuclear antibody (ANA) profiles in systemic lupus erythematosus (SLE). 94 patients with SLE, 70 patients with other rheumatic diseases and 30 healthy donors were examined. ANA (antibodies to doublestranded - dsDNA, Sm, SS-A/Ro, SS-B/La antigens, nucleosomes, ribosomal protein P-RibP and ribonucleoprotein - RNP-70) were determined in the serum by MIA using the xMAP technology. In MIA, antibodies to dsDNA, Sm and RibP have a high diagnostic specificity (Sp) (95.0-99.0%) and a likelihood ratio of positive results (LR+) (9.67-15.0), i.e. are the most "useful" diagnostic tests, and antibodies to RNP-70, SS-A/Ro and nucleosomes are classified as "useful" tests for the diagnosis of SLE (Sp: 84.0-95.0%, LR+> 2.0). Determination of profiles from 3 or more antigen-specific ANA by MIA increases the Sp method to 98.0-100%, and the LR+ - to the maximum values. Profiles from 7 subpopulations of ANA (antibodies to dsDNA, Sm, RibP, SS-A/Ro,SS-B/La, nucleosomes and RNP-70, 57.9%, 71.9%, 82.5%, 61.4 %, 84.2%, 50.9%, 84.2%) were found in the chronic variant of SLE. In the acute course of the disease, 4 subpopulations of ANA are simultaneously detected (antibodies to dsDNA, Sm, SS-A/Ro and nucleosomes, 77.3%, 45.5%, 40.9% and 72.7%); in subacute course there are 2 subpopulations of ANA (antibodies to dsDNA and nucleosomes, 53.3% and 46.7%). The activity index of SLEDAI-2K positively correlates with the concentration of antibodies to dsDNA (r = 0.55, p < 0.05), nucleosomes (r = 0.65, p < 0.05), RibP (r = 0.32; p < 0.05) and Sm (r = 0.36, p < 0.05) in the blood. There was no reliable relationship between the production of varieties of ANA and the index of organ damage. Mucocutaneous disorders, lupus-nephritis and neurolupus were most often associated with the detection of antibodies to dsDNA (53.2-64.0%), nucleosomes (55.3-66.0%), SS-A/Ro (38.0-40.4%) and Sm (27.8-36.2%). MIA of ANA profiles is an important tool for implementing a personalized approach to diagnosis, evaluation of activity, course and clinical and immunologic subtypes of SLE.

[The influence of STAT4 rs7574865 (G/T) polymorphism on the risk of clinical and immunological phenotypes of systemic sclerosis in a Russian patient population: Results of a pilot study]. / Vliianie polimorfizma rs7574865 (G/T) gena STAT4 na risk razvitiia klinicheskikh i immunologicheskikh fenotipov sistemnoi sklerodermii v russkoi populiatsii bol'nykh: rezul'taty pilotnogo issledovaniia.

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.

[THE CLINICAL INFORMATIVENESS OF AUTOMATED METHODS OF SCREENING DETECTION OF ANTI-NUCLEAR ANTIBODIES USING INDIRECT REACTION OF IMMUNE FLUORESCENCE, ENZYME-LINKED IMMUNOSORBENT ASSAY AND MULTIPLEX XMAP TECHNOLOGY UNDER SYSTEMIC LUPUS ERYTHEMATOSUS].

Thew antinuclear antibodies (ANA) consist heterogeneous group of auto antibodies reacting with various components of nucleus and cytoplasm. The ANA is a main serological marker of systemic lupus erythematosus (SLE). The implementation in clinical practice of new highly productive techniques of immune analysis using automated systems sets up prerequisites for standardization and amelioration of reproducibility of detection of ANA. The study was carried out to compare diagnostic significance of automated techniques of screening detection of ANA (indirect immunofluorescence test on cells HEp-2 (IIFT-HEp-2)), enzyme-linked immunosorbent assay (ELISA) and multi-complex immune analysis (MIA, using suspension technology xMAP) in serum of patients with SLE. The serums from 94 patients with SLE were analyzed. The comparison group included 70 patients with other rheumatic diseases. The control group consisted of 30 healthy donors. The screening detection of ANA using technique IIFT-HEp-2 was implemented on automated platform AKLIDES, ELISA - on automated analyzer ALEGRIA and MIA on automated analyzer BioPlex 2200. The technique IIFT-HEp-2 demonstrated the most high diagnostic sensitivity as compared with ELISA and MIA- BioPlex 2200 (96.8%; 79.8% and 82.9% correspondingly). The general diagnostic specificity of detection of ANA using technique IIFT-HEp-2 was lower than in case of ELISA and MIO-BioPlex 2200 (40%, 70% and 57% correspondingly). In the group of healthy donors the lowest diagnostic specificity was observed in ANA screening analysis using MIA-BioPlex 2200 (80%) while in case of applying IIFT-HEp-2 and ELISA indices of diagnostic specificity made up 93.3% and 96.7% correspondingly. The ANA analysis of mix of 26 nuclear antigens using ELISA technique was a reliable laboratory test for diagnostic of SLE (likelihood ratio of positive result - 2.66). By the level of likelihood ratio of negative result of the IIFT-HEp-2 technique was more informative test for exclusion of diagnosis of SLE than techniques of ELISA and MIA-BioPlex 2200 (0.08; 0.29 and 0.3 correspondingly). The detection of ANA using technique of is the most preferable primary screening test for diagnostic of SLE. The ELISA of antibodies to mix of nuclear antigens and MIA on the basis of xMAP technology are less preferable screening tests for diagnostic of SLE as compared with IIFT-HEp-2 because of false-negative results in 20% and 17% of cases correspondingly. ELISA and MIA are to applied as confirmatory screening tests permitting to detect antigen-specific ANA in patients with SLE with positive results of IIFT-HEp-2.

[Relationship between matrix metalloproteinase-3 levels and articular destructive changes in early and extended rheumatoid arthritis].

AIM: To estimate a relationship between matrix metalloproteinase-3 (MMP-3) levels and articular radiographic changes in early and extended rheumatoid arthritis (RA); to analyze the role of this biomarker in predicting the progression of joint destruction in RA. SUBJECTS AND METHODS: Forty-five patients with early RA and 42 with extended RA were examined. Radiography of the hands and distal feet was performed before and one year after therapy. Serum MMP-3 levels were measured by an enzyme immunoassay prior to and 12 and 24 weeks after treatment. RESULTS: After 52 weeks, in the early RA group, 16 patients continued monotherapy with methotrexate (MT); because of its inefficiency, 29 additionally received a biological agent in different follow-up periods. The extended RA group took tocilizumab for 24 weeks, then the drug was discontinued and the patients continued the former therapy with disease-modifying antirheumatic drugs, nonsteroidal anti-inflammatory drugs, and glucocorticosteroids. One year later, radiographic progression was recorded in 20.5 and 22.5% of the patients with early and extended RA, respectively. ROC analysis indicated that in the early RA group the MMP-3 level of more than 34.3 ng/ml at 12 weeks of MT therapy was associated with the radiographic progression of articular destructive changes after 52 weeks of therapy (the area under the curve (AUC) was 0.7; 95% confidence interval (CI) 0.46 to 0.93). In the patients with extended RA, the baseline MMP-3 levels of ≤51.3 ng/ml was related to no radiographic progression following 52 weeks (AUC, 0.587; 95% CI 0.33 to 0.84). CONCLUSION: MMP-3 may be regarded as an early marker for joint destruction in RA. The determination of MMP-3 level with other immunological markers may be useful to identify a group of patients who have a potentially severer disease course and need more intensive therapy.

[N-terminal pro-brain natriuretic peptide levels and diastolic dysfunction in patients with early rheumatoid arthritis before the administration of disease-modifying antirheumatic drugs].

AIM: To determine N-terminal pro-brain natriuretic peptide (NT-proBNP) levels in patients with early rheumatoid arthritis (RA) before the use of disease-modifying antirheumatic drugs (DMARDs); to compare NT-proBNP values with traditional risk factors (TRF), cardiovascular diseases (CVD), inflammatory markers, and left ventricular (LV) diastolic dysfunction (DD). SUBJECTS AND METHODS: The investigation enrolled 74 patients with a valid RA diagnosis (the 2010 ACR/EULAR criteria), 56 (74%) women, median (Me) age, 54 years; disease duration, 7 months; seropositive for IgM rheumatoid factor (87%) and/or anti-cyclic citrullinated peptide antibodies (100%) with no history of the use of DMARDs and glucocorticosteroids. Duplex scanning and echographic findings were used to assess TRF for CVD and carotid artery atherosclerosis (CAA) in all the patients with early RA prior to therapy. An E/A ratio was used as a criterion for LVDD. RESULTS: NT-proBNP concentrations in patients with early RA proved to be higher than those in the control group (p<0.0001). Higher-than-normal NT-proBNP levels were seen in 36 (49%) patients. The patients with early RA and elevated NT-proBNP values were older and had a higher body mass index (BMI) than those with normal NT-proBNP levels. Those with elevated NT-proBNP concentrations were more frequently found to have CAA, coronary calcification, and coronary heart disease; their intima-media thickness was also larger and C-reactive protein (CRP) levels higher than in those with normal NT-proBNP values. There were correlations between NT-proBNP levels and erythrocyte sedimentation rate, CRP, simplified disease activity index, and clinical disease activity index. Multivariate analysis revealed that chronic heart failure (CHF), CAA, CRP and low-density lipoprotein (LDL) levels, and BMI correlated with NT-proBNP concentrations. LVDD was detected in 35 (48%) patients with early RA. The level of NT-proBNP in patients with DD was higher than in those without DD. Higher-than-normal NT-proBNP values were observed in 23 (65%) and 12 (32%) patients with and without LVDD, respectively. The optimal NT-proBNP level for CHF detection was equal to 237.4 pg/ml (86% sensitivity and 85% specificity); the area under the ROC curve was 0.879. CONCLUSION: Just at the early disease stage, the patients are noted to have a high NT-proBNP level that is influenced by higher BMI, low LDL levels, CAA, CHF, and high CRP values. In the patients with early RA, the diagnostically significant NT-proBNP concentration for CHF detection was higher (237 pg/ml) than in those without RA (125 pg/ml). The patients with early RA should undergo NT-proBNP determination, LVDD screening, correction of TRF for CVD, atherosclerosis treatment, and remission achievement.

[THE IDENTIFICATION OF SUB-POPULATIONS OF B-LYMPHOCYTES OF PERIPHERAL BLOOD USING TECHNIQUE OF FLOW CYTOFLUOROMETRY IN HEALTHY INDIVIDUALS AND PATIENTS WITH RHEUMATOID DISEASES].

The study was carried out to apply technique of flow cytofluorometry for immunofenotyping of sub-populations of B-cells of peripheral blood in healthy persons and patients with rheumatoid diseases. The samples included 27 healthy donors, 16 patients with rheumatoid arthritis and 9 patients with systemic lupus erythematosus. The peripheral blood of all participants was analyzed for relative number of CD19+B-cells, total population of memory B-cells (CD19+CD27+); unswitched (CD19+IgD+CD27+) and switched (CD19+IgD- CD27+) memory B-cells, naive (CD19+IgD+CD27-) and transitory (CD19+IgD+CD10+CD38++CD27-) B-cells, plasmablasts (CD19+CD38+++IgD-CD27+CD20+) and long-lived plasmatic cells (CD19+CD138+). The sub-populations of B-cells were identified by technique of multicolor flow cytofluorometry using panel of monoclonal antibodies to surface membrane markers of B-lymphocytes. In normal state, relative number (median-Me; interquartile range 25-75 percentiles) of CD19+B-lymphocytes amounted to 9.1 (6.6-11.6)%; CD19+CD27+memory B-cells - 2.2 (1.6-3.3)%; unswitched and switched memory B-cells - 10 (6.4-12.7)% and 17.7 (14.9-27.0)%; naive B-cells - 65.8 (55.1-73.4)%; transitory B-cells - 0.1 (0.1-0.3)%; plasmablasts - 7.0 (5.0-9.4)%. The long-lived plasmatic cells in peripheral blood were absent. In patients with rheumatoid arthritis percentage of content of total population of memory B-cells were lower than in donors (1.6; 1.00-2.3%; p = 0.038). Under systemic lupus erythematosus was detected decreasing of number of naive B-cells (40.2; 19.7-58.2) and increasing of level of switched memory B-cells (34.2; 21.0-52.7) as compared with donors (p = 0.003 in both cases). The study established no reliable differences between healthy donors and patients with rheumatoid arthritis and systemic lupus erythematosus in the rest of sub-populations of B-lymphocytes. The developed technique of multi-parametric flow cytofluorometry can be recommended for studying homeostasis of B-cells of peripheral blood in healthy persons and patients with auto-immune rheumatoid diseases and also for evaluating and forecasting effectiveness of anti-B-cells therapy.

[Autoimmune Rheumatic Diseases - Problems of Immunopathology and Personalized Treatment].

By current standards autoimmunity is a complex pathological process based on a violation of tolerance and, consequently, the pathological immune response against its own tissues components (autoantigens) leading to the development of a wide range of autoimmune diseases in humans. In recent years, multiple immune disorders both acquired and/ or congenital (associated with polymorphisms of genes that regulate immune response) have been transcribed. These disorders occur at the cellular and humoral levels: thymus, intestines, peripheral blood immune cells, including T and B lymphocytes, macrophages, dendritic cells, Treg-cells (Treg), components of complement system, cytokines and others. The interaction between the development of autoimmune rheumatic (ARD) and autoinflammatory diseases and syndromes is detected; a classification of immune-inflammatory diseases is designed. The article describes the results of our studies on the treatment of ARD using innovative genetically engineered biological agents and on the research ofpathogenetic mechanisms and diagnostics of ARD based on immunological and molecular biological diagnostic techniques of a wide range of molecular and cellular biomarkers (autoantibodies, inflammatory acute phase proteins, cytokines, chemokines, markers of activation of the vascular endothelium, the components of the complement system, lymphocyte subpopulations, products of metabolism of bone and cartilage tissue, genetic, epigenetic, transcriptomic markers). The approaches to personalized treatment of ARD are presented.

[The automated analysis of anti-nuclear antibodies using technique of indirect reaction of immunofluorescence with application of HEP-2-cells].

The identification of antinuclear antibodies in blood serum based on indirect reaction of immunofluorescence using cells of line HEp-2 (IRIF HEp-2)--a "golden standard" and key screening technique of laboratory diagnostic of autoimmune rheumatic diseases. The automated systems of interpretation of samples offluorescence promote standardization and increase effectiveness of detection of content of antinuclear antibodies with IRIF HEp-2 technique. The study was organized to comparatively analyze automated and visual interpretation of results of IRIF HEp-2 in detection of content antinuclear antibodies in patients with rheumatic diseases. The level of antinuclear antibodies in blood serums of 1178 patients with rheumatic diseases was detected using IRIF HEp-2 technique. The results of IRIF HEp-2 were evaluated by visual microscopy and using automated platform "AKLIDES". The degree of consistency of positive/negative results of detection (k = 0.5), types (k = 0.7) and titers/intensity of fluorescence (k = 0.45) of antinuclear antibodies under automated and traditional interpretation of IRIF HEp-2 was "good". The discordance of positive/negative results of analysis of content of IRIF HEp-2 was established in 18.5% of patients. The automated technique more often detected homogeneous (37.6%) and speckled (32.3%) fluorescence of nucleus. At the same time, there were no differentiation of type of fluorescence in 21.4% of patients. The visual technique detected mixed type of fluorescence in blood serums of most of the patients (72.8%). The mixed fluorescence was identified by system "AKLIDES" as homogeneous (40.5%), speckled (32.7%), nucleolar (2.4%), centromeric (0.9%), undifferentiated (23.5%). Under visual analysis of samples of fluorescence with undifferentiated type of fluorescence was identified as mixed (79.8%), homogeneous (5.9%) and speckled (14.3%). The titers of antinuclear antibodies less than 1:160 associated with intensity of fluorescence 0/B±; 1:160-0, B±, +, ++; more than 1:1280--+++, ++++. In common practice the automated system "AKLIDES" permits identifying positive/negative results of detection of content of antinuclear antibodies comparably with "classic" visual technique of interpretation of IRIF HEp-2 and prognosticate maximal finite titer of antinuclear antibodies in serums in patients with rheumatic diseases according intensity of fluorescence. To confirm results of automated evaluation of types of nuclear fluorescence and to specify titers of antinuclear antibodies it is recommended to apply additional expert visual analysis of positive samples of fluorescence.

[IgG4-related salivary gland lesions]. / Svyazannye s IgG-4 porazheniya slyunnykh zhelez.

AIM: To provide the demographic, clinical, laboratory, radiological, morphological, and immunomorphological characteristics of IgG4-related sialoadenitis (IgG4-S), which allow the differential diagnosis with neuroendocrine, granulomatous, blood cancer lesions of the salivary gland (SG). SUBJECTS AND METHODS: In the period 2004 to 2014, IgG4-S was diagnosed in 32 (11%) out of 289 patients with significantly enlarged parotid and submandibular glands (PG and SMG). Only 4 (9%) patients had isolated IgG4-related disease (IgG4-D) whereas involvement of a few organs ran as an IgG4-SD systemic disease in 29 (91%) patients. RESULTS: There was a slight preponderance of women with a median onset age of 42 years. Enlargement of the SMG (52.7%), lesions of the nasal cavity and paranasal sinuses (37.2%), and enlargement of the lacrimal gland and orbital pseudotumors (31%) are the most common clinical manifestations at disease onset. A follow-up study indicated that along with involvements of SMG (97%), PG (72%), eye sockets (72%), nasal cavity and paranasal sinuses (56%), one third of the patients were found to have generalized lymphadenopathy, to frequently develop pulmonary, hepatic, pancreatic, renal injuries; and the disease ran within IgG4-SD. The laboratory manifestations were characterized by moderate eosinophilia and elevated blood IgE levels in one-third of the patients and by moderately higher erythrocyte sedimentation rate and hypergammaglobulinemia in 50%. The increased blood level of IgG (84%) and its subclass IgG4 (86.4%) is an indication for further verification of IgG4-D in patients with involvement of the major SG. Immunohistochemical examination, by measuring the concentration of IgG4-secreting plasma cells (PCs), and determination of B-cell clonality in biopsy specimens should be done to verify a diagnosis with IgG4-D. CONCLUSION: The determination of blood IgG4 (>2 g/l) in patients with considerably enlarged major SG may suggest the presence of IgG4-S. Minimally invasively incised PG and SMG biopsies with their subsequent morphological and immunomorphological examinations should be performed to make an accurate diagnosis. More than 40% of IgG4-secreting PCs detected in SG tissue is evidence to diagnose IgG4-D.

[The importance of cytokine profile characteristics for evaluating the therapeutic effectiveness of monoclonal antibodies against IL-6 receptors in patients with rheumatoid arthritis].

AIM: To evaluate dynamics of cytokine profile characteristics in patients with rheumatoid arthritis (RA) treated with tocilizumab and to identify parameters that can be used to predict the effectiveness of therapy. MATERIALS AND METHODS: 42 patients (32 women) aged 43-55 (mean 50.5) years with the duration of disease 23-81 (mean 56.5 months), DAS28 6.4 (5.8-7.05). Each patient was given 6 i/v infusions of 8 mg tocilizumab/day at 4 week intervals in addition to standard therapy. Serum levels of IL-1b, IL-1Pa, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF-basic, G-CSF, GM-CSF, IFN-g, IP-10, MCP-1, MIP-1alpha, MIP-1b, PDGF bb, RANTES, TNF-alpha, VEGF were determined by xMAP multiplex technology. RESULTS: Good therapeutic effect in accordance with EULAR criteria was documented in 35 and satisfactory one in 7 patients; remission based on CDAI occurred in 33% of the cases. The levels of proinflammatory (IL-1b, -2, -6, -12, -15, -17, IFN-gamma, TNF-alpha) and anti-inflammatory (IL-4, -5, -9, -10, -13) cytokines, hemokines (IL-8, MCP-1, MIP-1a, MIP-1b, MIP-1b) and growth factors (IL-7, GM-CSF, VEGF, FGF basic, IP-10) dropped down by week 24 of the treatment (p < 0.05). Remission based on CDAI was associated with higher baseline levels of IL-1b, -2, GM-CSF and TNF-alpha and good outcome according to EULAR criteria with the rapid fall in IL-10 and -13 levels. CONCLUSION: Therapy with tocilizumab results in the rapid and well apparent decrease in the concentration of the practically entire spectrum of cytokines. Measurement of IL-1b, -2, -10, -13, GM-CSF and TNF-alpha may be useful for the prediction of the effectiveness of therapy of RA with tocilizumab.

[Evaluation of methotrexate effect on the acute-phase response in rheumatoid arthritis after 12-week treatment].

UNLABELLED: DAS28 index calculated with regard for ESR, the number of swollen/painful joints and evaluation of the patient's condition by VAS is universally used to estimate activity of rheumatoid arthritis (RA). There is a variant of calculation using C-reactive protein (CRP) instead of ESR. Our experience indicates that ESR decreases more slowly than CRP during treatment and better reflects dynamics of patients' condition. From the practical standpoint it is important to estimate activity of RA because therapeutic modalities are chosen based on the DAS28 value. AIM: To study the influence of pharmaceutical form of methotrexate on the acute-phase response in rheumatoid arthritis. MATERIALS AND METHODS: The study included 32 patients (24 women, 8 men) aged 19-76 (mean 47.5 +/- 28.5) yr with active RA (DAS28 > 3.2) 4-30 months (11.5 +/- 7.4, median 8) in duration. Diagnosis was made using AXR criteria (1987), none of the patients previously received methotrexate injections. Inclusion criteria: initially high ESR (Westegren, mm/hr) and/or CRP (mg/l measured by a highly sensitive method). All patients were given methotrexate subcutaneously for 12 weeks as monotherapy (initial dose 10 mg, maximum one 25 mg/week). The cumulative dose was 211.36 +/- 17.2 mg. RESULTS: Side effects did not require withdrawal of methotrexate. CRP level decreased faster than ERS: a 70% decrease of CRP by week 12 was recorded more frequently than that of ESR. Slow dynamics of the number of swollen joints compared with CRP may be due to the low cumulative dose of methotrexate. Duration of the disease had no effect on dynamics of acute phase characteristics. CONCLUSION: Methotrexate injections resulted in markedly delayed development of clinical signs of improvement compared with laboratory values. CFP levels fell down much faster than ESR, Remission or low activity of RA (estimated from DAS28) occurred only in 38% of the cases after 3 month monotherapy by methotrexate injections. It is concluded that efficacy of this drug should be estimated no sooner than 4 months after the onset of the treatment.

[The immunologic predictors of effect of anti-B-cell therapy under rheumatoid arthritis].

The article deals with results of study targeted to reveal laboratory biomarkers which can be useful in prognosis of effectiveness of rituximab therapy under rheumatoid arthritis. The sampling consisted of 34 patients with rheumatoid arthritis (31 women, average age 49 years, 42-64 years, mean duration of disease 66, 36-132 months). All patients were examined and received two infusions of rituximab intravenously with interval in 2 weeks against the background of standard therapy. The serum concentration of C-reactive protein, IgM rheumatoid factor IgG, IgM, IgA were measured using immune nephelometric method. The level of cyclic citrullinated peptide antibodies, modified citrullinated vimentin antibodies and IgA rheumatoid factor was measured using method of immune enzyme analysis. The panel of 27 cytokines was measured using multiplex technology xMAP. Before rituximab therapy indices DAS28 (6,12; 5. 52-6, 81), SDA1 (34.3; 23, 8-45, 9) and CDAI (31.3; 21, 8-38.5) corresponded to high activity of rheumatoid arthritis. Up to 24th week of therapy good response on criteria EULAR was registered in 15 patients, moderate response in 18 patients and was absent in 1 patient. The remission on DAS achieved more rarely in patients with initially negative/ low positive values of IgM rheumatoid factor, basal level of IgM less than 2.4 g/l and duration of disease more than 40 months. In the group of patients who attained remission on CDAI up to 24th week of therapy higher basal level of IL-IRA, IL-2, IL-8, IL-15, Eotaxin, GM-CSF, IFN-gamma, MIP-1alpha and TNF-alpha was registered In patients who attained remission on DAS 28 higher level of IL-1beta. IL-2, IL-6, G-CSF, IFN-gamma, MIP-1alpha and TNF-alpha was registered in comparison with patients with disease in active mode. The detection of basal level of IgM rheumatoid factor, IgM and also certain cytokines (IL-1beta, IL-IRA, IL-2, IL-8, IL-15, GM-CSF, IFN-gamma, MIP-1alpha, Eotaxin, TNF-alpha) can be useful in prognosis of effectiveness of rituximab therapy under rheumatoid arthritis.