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.

[Zonulin and I-FABP are markers of enterocyte damage in celiac disease].

AIM: To evaluate the level of serum I-FABP (Fatty-Acid-Binding Protein a protein that binds fatty acids) and fecal zonulin as markers of the permeability of the mucous membrane of the small intestine in celiac patients. MATERIALS AND METHODS: A total of 151 celiac patients (25 men and 126 women) were examined. The median age was 42 years. Group I included 58 patients with newly diagnosed celiac disease; in group 2 38 patients, knowingly or unknowingly violating the gluten-free diet; group 3 consisted of 55 patients strictly observing gluten-free diet. The control group consisted of 20 healthy volunteers: 4 men and 16 women. All patients underwent esophagogastroduodenoscopy by biopsy of the mucous membrane of the small intestine and assessment of duodenobioptates according to Marsh. In the blood serum, the level of antibodies to tissue transglutaminase IgA and IgG was determined by the enzyme-linked immunosorbent assay using kits manufactured by Orgentec Diagnostics GmbH (Germany), the concentration of I-FABP in blood serum was determined using Hycult Biotech kits (Netherlands). The content of zonulin in feces was investigated by enzyme-linked immunosorbent assay using kits from Immundiagnostik AG (Germany). Statistical analysis was performed using the Statistica 13.3 software (StatSoft Inc., USA). RESULTS: There was a significant increase in the level of antibodies to tissue transglutaminase IgA [120.0 (41.1200)] IU/ml and IgG [31.4 (5.578.9)] IU/ml in patients of group 1 compared with group 2 [IgA 9.1 (2.987.6)] and IgG [3.8 (2.219.7)] IU/ml and group 3 [IgA 1.6 (1.03.2)] and IgG [2.2 (1.152.53)] (p0.01). The level of I-FABP in blood serum in patients of group 1 averaged 2045 pg/ml, in patients in group 2 1406 pg/ml, in patients in group 3 1000 pg/ml. All patients showed a significant increase in the mean I-FABP values compared to controls (1, 2 and control p0.01, 3 and control p=0.016). In patients with Marsh grade III AC atrophy, the I-FABP level depended on the degree of damage to the mucosa and significantly differed from the control: March IIIA (median: 1310 pg/ml, interquartile range: 12121461 pg/ml), March IIIB (median: 2090 pg/ml, interquartile range: 18122322 pg/ml) as well as Marsh IIIC (median: 2058 pg/ml, interquartile range 18582678 pg/ml). The concentration of zonulin in feces in patients of group 1 averaged 111.6 pg/mg, in patients of group 2 90.5 pg/mg. In patients of group 3 50 IU/ml. The concentration of zonulin in feces increased as the degree of mucosa atrophy increased (r=0.585, p0.01). However, despite the fact that both of these markers may indicate impaired permeability, each of them indicates damage to a certain level of the intestinal barrier, which is not always associated with the degree of mucosa atrophy. CONCLUSION: Determination of serum I-FABP and fecal zonulin levels in celiac patients allows for the assessment of intestinal permeability and can serve as non-invasive markers for monitoring ongoing structural changes in the mucosa without the need for endoscopy.

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.

Anaerotalea alkaliphila gen. nov., sp. nov., an alkaliphilic, anaerobic, fermentative bacterium isolated from a terrestrial mud volcano.

Diversity of extremophilic microorganisms in mud volcanoes is largely unexplored. Here, we report the isolation of a novel alkaliphilic, mesophilic, fermentative bacterium (strain F-3apT) from a terrestrial mud volcano located at the Taman peninsula, Russia. Cells of strain F-3apT are Gram-stain-positive non-motile rods. The formation of endospores is not observed. The temperature range for growth is 14-42 °C, with an optimum at 37 °C. The pH range for growth is 7.5-11.0, with an optimum at pH 9.0. The isolate utilizes various organic polymeric substances, organic acids, carbohydrates, and proteinaceous compounds. The end products of glucose fermentation are ethanol, CO2, and H2. The major cellular fatty acids of strain F-3apT are C16:0, C16:1, and C14:0. Phylogenetic analysis reveals that strain F-3apT belongs to the order Clostridiales, with less than 91% of 16S rRNA gene sequence similarity to any species with a validly published name. The total size of the genome of strain F-3apT is 2.98 Mb, and a genomic DNA G + C content is 56.78 mol%. The whole-genome phylogenetic analysis confirms that strain F-3apT forms a distinct lineage within Clostridia. We propose to assign strain F-3apT to a new species of a novel genus Anaerotalea alkaliphila gen. nov., sp. nov. The type strain is F-3apT (= KCTC 15917 T = VKM B-3406 T).

[Enteropathy with impaired membrane digestion and the prospects for cytoprotective therapy].

The article describes enteropathy with impaired membrane digestion (EIMD) as a new nosological form. The main clinical manifestation of EIMD is the poor tolerance of food products, in particular carbohydrates and a decrease in the activity of membrane enzymes, in particular, carbohydrates, in the mucous membrane of the small intestine. The cause of the disease can be acute intestinal infections, viruses, drugs and other agents that damage the small intestine. The pathophysiology, clinical picture and diagnosis of EIMD are described. The basis of therapy is rebamipide, which has the ability to reduce the symptoms of carbohydrate intolerance and increase the activity of disaccharidases.

[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.

Pelomicrobium methylotrophicum gen. nov., sp. nov. a moderately thermophilic, facultatively anaerobic, lithoautotrophic and methylotrophic bacterium isolated from a terrestrial mud volcano.

A novel moderately thermophilic, bacterium, strain SM250T, was isolated from a terrestrial mud volcano, Taman peninsula, Krasnodar region, Russia. Cells of strain SM250T were Gram-negative non-spore forming motile straight rods. Growth was observed at temperatures 30-63 °C (optimum at 50 °C), pH 6.5-10.0 (optimum at pH 8.5) and NaCl concentrations 0-4.5% (w/v) (optimum at 1.0-1.5% (w/v)). The novel isolate grows by aerobic respiration or anaerobic respiration with nitrate as the terminal electron acceptor. Strain SM250T grows by the utilization of methanol, formate and a number of other organic compounds or lithoautotrophically with hydrogen, elemental sulfur or thiosulfate as electron donors. The total size of the genome of the novel isolate was 3,327,116 bp and a genomic DNA G + C content was 64.8 mol%. Analysis of the 16S rRNA gene sequences revealed that strain SM250T belongs to the class Hydrogenophilia within the phylum Proteobacteria, with less than 91% of 16S rRNA gene sequence similarity to any species with validly published name. We propose to assign strain SM250T to a new species of a novel genus Pelomicrobium methylotrophicum gen. nov., sp. nov. The type strain is SM250T (= KCTC 62861T = VKM B-3274T).

[Dual-energy computed tomography in the diagnostics of urolithiasis].

THE AIM: To assess the diagnostic performance of dual-energy computed tomography (DECT) in the evaluation of the composition of urinary stones "in vivo". MATERIALS AND METHODS: A total of 91 patients aged from 20 to 70 years old (mean 42.7) with urinary stone disease were examined at Sechenov University, including 68 men (75%) and 23 women (25%). Prior to surgery, all patients underwent DECT (Canon, Japan) in order to predict the chemical composition of urinary stones in vivo. Extracorporeal shockwave lithotripsy (ESWL), percutaneous nephrolithotomy (PCNL) and ureteroscopy (URS) was performed in 53 (58.2%), 18 (19.7%) and 20 (22.1%) patients, respectively. Postoperatively, all stones or stone fragments (n=91; 100%) were examined using a comprehensive physical and chemical analysis (X-ray phase analysis, electron microscopy, infrared spectroscopy). RESULTS: In 6 patients (6.6%) staghorn stones were diagnosed, while in 15 (16.5%), 17 (18.7%), 22 (24.2%) and 31 (34.1%) stones were located in ureteropelvic junction, pelvis and ureter, respectively, including 24 patients with lower ureteric stones (26.4%). Prediction of the stone composition in vivo was carried out on the basis of the one indicator, the dual energy ratio (DER). The threshold values of DER for different types of stones were taken from the literature. All stones were divided into 4 groups according to the DECT results: vevellite stones (n=40, 43.9%), Ca-containing stones without vevellite (n=34, 37.3%), uric acid stones (n=10, 10.9%) and struvite stones (n=7, 7.9%). Thus, when comparing the results of DECT and physical and chemical analysis, in the first group four stones were incorrectly assigned by DECT to the group of Ca-containing stones without vevellite and three stones were incorrectly assigned to the group of struvite stones; in the second group four stones were incorrectly assigned to the group of vevellite stones; in the third group one stone was incorrectly assigned to the group of struvite stones; in the fourth group one stone was incorrectly assigned to the group of vevellite stones and one stone in the group of uric acid stones. In order to increase the diagnostic efficiency of DECT, we performed a comprehensive analysis of five specific DECT indicators (stone density at 135 kV, Z eff of the stone, DER, DEI, DED) using discriminant analysis. Thus, the sensitivity, specificity and overall accuracy of DECT with the use of just one indicator (DER) were 83.3%, 89.8%, 86.8% for vevellite, 88.2%, 92.9%, 91.2% for Ca-containing stones without vevellite, 90%, 98.8%, 97.8% for uric acid stones and 60%, 95.3%, 93.4% for struvite stones, respectively. When using discriminant analysis with five specific DECT indicators, higher values of sensitivity, specificity and overall accuracy were seen: 95.2%, 89.8%, 92.3% for a vevellite, 85,3%, 96,4%, 92,3% for Ca-containing stones without a vevellite and 100%, 100% and 100% for both uric acid and struvite stones, respectively. CONCLUSIONS: Dual-energy computed tomography is a highly informative method which allows to perform preoperatively the reliable assessment of the chemical composition. DECT in patients with urinary stone disease allows to optimize the treatment strategy and carry out preventive measures on individual basis, taking into account the stone type.

Study of acoustic and low-pressure exposure on the temperature of the evaporation of a liquid with free interface before it freezes.

On the basis of the first law of thermodynamics, a thermodynamic physical and mathematical model of the process of evaporation and freezing in a partially filled closed volume of a liquid under acoustic exposure (AE) and pressure reduction has been developed. The acoustic effect on the liquid is taken as a component on the heating of the liquid in the closed volume. The experimental studies performed under AE (amplitude 2.0 µm, frequency 25 kHz), initial temperature, and mass of the liquid 300.85 K, 7 g, respectively, the pressure decreases from 101 to 0.5 kPa showed a close coincidence of the actual and calculated moments of freezing start liquid surfaces, as well as close calculated and experimental values of liquid temperatures during the experiment.

Thioflexithrix psekupsensis gen. nov., sp. nov., a filamentous gliding sulfur bacterium from the family Beggiatoaceae.

A sulfur-oxidizing, filamentous, gliding micro-organism, strain D3T, was isolated from a sulfidic spring in Goryachy Klyuch, Krasnodar, Russia. The cell walls were Gram-negative. The new isolate was a microaerophilic facultative anaerobe and an obligate chemolithoautotroph. The pH range for growth was pH 6.8-7.6, with an optimum at pH 7.2. The temperature range for growth was 10-46 °C, with an optimum at 32 °C. The G+C content of DNA was 42.1 mol%. Phylogenetic analysis of the 16S rRNA gene showed that strain D3T belongs to the family Beggiatoaceae, order Thiotrichales and was distantly related to the genera of the family Beggiatoaceae(86-88 % sequence similarity). The major respiratory quinone was ubiquinone-6. Major fatty acids were C18:1 ω7 (37.6 %), C16 : 0 (34.7 %) and C16: 1 ω7 (27.7 %). On the basis of its physiological properties and the results of phylogenetic analysis, strain D3T is considered to represent a novel species of a new genus, for which the name Thioflexithrix psekupsensis gen. nov., sp. nov. is proposed. The type strain is D3T (=KCTC 62399=UNIQEM U981).

[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.

Amplification of surface-enhanced Raman scattering by the oxidation of capping agents on gold nanoparticles.

Surface-enhanced Raman spectroscopy is a powerful analytical method, and is especially useful for the detection of nitrogen- and sulfur-containing organic substances in trace amounts. SERS substrates with high enhancement factors can be produced via the aggregation of gold nanoparticles, leading to the formation of 'hot spots' - regions of highest electric field intensity and Raman scattering enhancement. Thus, the availability of gold surfaces in 'hot spots' for the adsorption of analyte molecules strongly influences the enhancement factor of a substrate. We studied the kinetics of oxidation of dyes with hydrogen peroxide in the presence of citrate-capped gold nanoparticles and discovered the amplification of surface-enhanced Raman scattering, probably due to the oxidation of citrate ligands and the additional adsorption of dye molecules onto vacant spots on the gold surface. Maximum amplification was observed with 3% (v/v) hydrogen peroxide in the reaction medium. Under optimized conditions, model analytes can be detected at concentrations as low as 1 × 10-9 M, which is ten times lower than the detection limit without hydrogen peroxide addition.

[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.

Thermodesulfitimonas autotrophica gen. nov., sp. nov., a thermophilic, obligate sulfite-reducing bacterium isolated from a terrestrial hot spring.

A novel thermophilic, anaerobic, chemolithoautotrophic bacterium, strain SF97T, was isolated from a terrestrial hot spring (Kuril Islands, Russia). Cells of strain SF97T were rod-shaped and motile with a Gram-positive cell-wall type. The novel isolate grew at 45-72 °C (optimum 65 °C) and pH 5.5-8.5 (optimum 6.0-6.5). The strain grew chemolithoautotrophically with molecular hydrogen as an electron donor, sodium sulfite or SO2 gas as an electron acceptor and bicarbonate/CO2 as a carbon source. Sulfate, thiosulfate, elemental sulfur, Fe(III) or nitrate were not used as electron acceptors either with H2 or organic electron donors. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the family Thermoanaerobacteraceae, order Thermoanaerobacterales, and was distantly related to species of the genus Ammonifex (92-93 % sequence similarity). On the basis of its physiological properties and results of phylogenetic analyses, strain SF97T is considered to represent a novel species of a new genus, for which the name Thermodesulfitimonas autotrophica gen. nov., sp. nov. is proposed. The type strain of Thermodesulfitimonas autotrophica is SF97T (=DSM 102936T=VKM B-2961T). T. autotrophica is the first reported obligate sulfite-reducing micro-organism.

[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.

[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.

Dissulfurimicrobium hydrothermale gen. nov., sp. nov., a thermophilic, autotrophic, sulfur-disproportionating deltaproteobacterium isolated from a hydrothermal pond.

A thermophilic, anaerobic, chemolithoautotrophic bacterium (strain Sh68T) was isolated from a hydrothermal pond at Uzon Caldera, Kamchatka, Russia, using anoxic medium with elemental sulfur as the only energy source. Cells of strain Sh68T were Gram-stain-negative rods, 0.5-0.8 µm in diameter and 1.2-2.0 µm in length, motile by means of flagella. The temperature range for growth was 30-65 °C, with an optimum at 50-52 °C. The pH range for growth was 5.2-7.5, with optimum growth at pH 6.0-6.2. Growth of strain Sh68T was observed at NaCl concentrations ranging from 0 to 2.3 % (w/v). Strain Sh68T grew anaerobically with elemental sulfur as an energy source and bicarbonate/CO2 as a carbon source. Elemental sulfur was disproportionated to sulfide and sulfate. Growth was enhanced in the presence of poorly crystalline Fe(III) oxide (ferrihydrite) as a sulfide-scavenging agent. Strain Sh68T was also able to grow by disproportionation of thiosulfate and sulfite. Sulfate was not used as an electron acceptor either with H2 or with organic electron donors. Analysis of the 16S rRNA gene sequence revealed that the isolate belongs to the class Deltaproteobacteria and is related most closely to Dissulfuribacter thermophilus S69T (90.0 % similarity). On the basis of its physiological properties and results of phylogenetic analyses, strain Sh68T is considered to represent a novel species of a new genus, for which the name Dissulfurimicrobium hydrothermale gen. nov., sp. nov. is proposed. The type strain of Dissulfurimicrobium hydrothermale is Sh68T ( = JCM 19990T = VKM B-2854T). This is the first description of a sulfur-disproportionating thermophile from a terrestrial ecosystem.