B Cell Profiling in Patients with Pemphigus Vulgaris.

Pemphigus vulgaris is a severe, socially significant autoimmune disease associated with autoantibodies to the desmoglein 3 antigen. The disease affects all age groups, beginning at 18 years of age; the mortality rate of pemphigus can reach as high as 50%, depending on a patient's age and a number of other factors. There is no highly selective or personalized therapy for pemphigus vulgaris at the moment. One of the well-known therapeutic approaches to the disease is to use rituximab, an anti-CD20 antibody that can help achieve B cell depletion in peripheral blood. To solve the problem of nonspecific elimination of B cells in patients with pemphigus vulgaris, it is reasonable to use specific immunoligands, their choice being based on an assessment of the level of autoantibodies specific to each of the fragments of desmoglein. In this work, the proportion of autoreactive B cells in patients diagnosed with pemphigus vulgaris is found to be 0.09-0.16%; a positive correlation was revealed between the antibody level and the number of autoreactive B cells to various fragments of desmoglein.

Analysis of the Specificity of Auto-Reactive Antibodies to Individual Fragments of the Extracellular Domain of Desmoglein 3 in Patients with Pemphigus Vulgaris.

A method for the analysis of the epitope specificity of auto-reactive antibodies to desmoglein 3 (Dsg3) using competitive ELISA has been developed. It is based on a two-stage solid-phase ELISA with initial "depletion" of auto-reactive antibodies against the studied epitope and subsequent quantitative assessment of antibodies against full-length extracellular domain Dsg3. The proposed approach for assessing the specificity of the autoimmune response in patients with pemphigus vulgaris can provide in the future the possibility to personalize the therapy using plasmapheresis by preliminary selection of the antigenic composition of the extracorporeal immunosorbent.

Recombinant Fragment of the Extracellular Domain of Human Desmoglein 3 Fused with the Fc-Fragment of Human IgG1 Selectively Adsorbs Autoreactive Antibodies from the Sera of Pemphigus Patients.

Using the recombinant second fragment of the extracellular domain (EC2) of human desmoglein type 3 (Dsg3) as an affinity ligand, an immunosorbent was obtained that selectively binds autoreactive antibodies to this domain from the immune sera of patients with pemphigus. The EC2 protein was obtained in the form of a fusion protein with the Fc-fragment of human IgG1. The production was carried out in CHO cells using the method of transient expression.

Experimental Modeling of Leprosy in BALB/c, BALB/c Nude, CBA, and C57BL/6ТNF-/- Mice.

Leprosy was modeled in an experiment on BALB/c, BALB/cNude, CBA, and C57BL/6ТNF-/- mice using three Mycobacterium leprae strains obtained from patients with a diagnosis of A30 according to ICD-10 from different regions of the Russian Federation. Proliferation of M. leprae of the used strains showed a temporal-quantitative dependence on the used mouse line. CBA and BALB/cNude mice were optimal for strain R and BALB/c and BALB/cNude lines were optimal for strain I. BALB/cNude mice infected with strain I had low lifespan. M. leprae strain M showed low proliferation activity in BALB/cNude and C57BL/6ТNF-/- mice.

Whole-Genome Sequencing of Russian Neisseria Gonorrhoeae Isolates Related to ST 1407 Genogroup.

The whole-genome sequencing data of three N. gonorrhoeae strains isolated in the Russian Federation in 2015 are presented. According to the NG-MAST protocol, these strains are related to the globally spread ST 1407 genogroup. The analysis of their resistomes showed the absence of ermA/B/C/F genes and the presence of wild-type alleles of rpsE, rrs, rrl, rplD, rplV, macAB, and mefA genes, and these patterns explain the susceptibility of the sequenced strains to aminocyclitols (spectinomycin) and macrolides (azithromycin). Conjugative resistance determinants (blaTEM, tetM) were absent in the genomes, and the penC/ pilQ, parE, and norM alleles were shown to be wild-type, whereas single or multiple nucleotide substitutions were identified in the genes encoding targets for ß-lactams (ponA, penA), tetracyclines (rpsJ), and fluoroquinolones (gyrA, parC). The additional mutations were found in porB gene and the promoter of mtrR gene, which nonspecifically reduced the susceptibility to antimicrobials due to the membrane permeability decrease and efflux pump overexpression. The diversity of mutations observed in the analyzed genomes prompted a revision of the phylogenetic relationships between the strains by comparing more than 790 groups of housekeeping genes. A high homology between the N. gonorrhoeae ST 1407 and N. gonorrhoeae ST 12556 genomes was confirmed; the latter had probably diverged from a common ancestor as a result of single mutation events. On the other hand, N. gonorrhoeae ST 12450 was an example of phenotypic convergence which appeared in the emergence of new drug resistance determinants that partially coincide with those of the ST 1407 genogroup.

Immunochip for Syphilis Serodiagnostics with the Use of Extended Array of Treponema pallidum Recombinant Antigens.

An immunochip for multiple parallel detection of specific serum IgG in serological screening for syphilis is based on the use of an extended array of Treponema pallidum recombinant proteins and includes traditionally used immunodominant antigens (Tp15, Tp17, Tp47, and TmpA) and new synthetic proteins (Tp0277, Tp0319, Tp0453, Tp0684, Tp0965, and Tp1038). The use of individual antigens has demonstrated high analytical value of Tp0277 (periplasmatic C-terminal protease), Tp0319 (cytoplasmic membrane-associated lipoprotein TmpC), and external membrane-associated protein Tp0453 with transporting function, all of them improving significantly the efficiency of screening for syphilis in comparison with the traditional array of antigens. Multiparametric analysis of the results obtained on the immunochip with the use of linear discriminant analysis confirmed the efficiency of extended array of T. pallidum diagnostic antigens. Due to proposed modification, the "positive" and "negative" sera are clearly differentiated: the serological study showed 94.1% sensitivity and 100% specificity.

[The refinement of leprosy PCR diagnostics by the amplification of specie-specific repeated fragment of the Mycobacterium leprae genome.]

Certain level of new registered cases of leprosy in a number of endemic countries in the world, as well as growing rate of transboundary migratory flows, raise the issue of effective diagnosis of this disease in countries with sporadic incidence of leprosy, including the Russian Federation. The purpose of the study was to develop a highly sensitive PCR test for detecting the genetic material of Mycobacterium leprae and to compare the test robustness and sensitivity with the commercially available Leprosy Genesig Standard Kit (Primerdesign Ltd., UK). The proposed approach uses real time PCR of non-coding repeating element RLEP, unique for the M. leprae genome, using TaqMan probe. The high test specificity was shown using the reference DNA samples of pathogenic and conditionally pathogenic mycobacterium, as well and its comparison with single-copy genes of M. leprae (rrs, fbp, MntH) PCR detection. The use of a commercially available test system based on the single-copy rpoB gene detection provided 59.4% sensitivity to the detection of M. leprae in the clinical material, while the application of the developed approach increased this index to 96.8%. The developed PCR diagnostics test of leprosy is submitted for state clinical approval process, whereupon the practical use of the test diagnostics allows solving a wide range of tasks to identify and confirm new cases of leprosy, and monitoring both the effectiveness of leprosy treatment, and epidemiological (including transboundary) the spread of the disease.

Oligonucleotide Microchip for the Identification of Infectious Agents of Reproductive System with Simultaneous Analysis of Determinants of Resistance to Antimicrobial Substances.

We developed a multiplexed DNA microarray-based assay allowing identification of 12 causative agents of reproductive tract infections with the simultaneous detection of 47 genetic determinants of resistance to antimicrobial substances. The microarray was tested on 93 isolates of Neisseria gonorrhoeae, 32 isolates of Treponema pallidum and 29 samples of Ureaplasma spp./Mycoplasma spp. The N. gonorrhoeae isolates had multiple mutations in the penA, ponA, rpsJ, gyrA, parC, and mtrR genes; their prognostic value significantly increased when combinations of mutations were detected. In the analyzed T. pallidum isolates, single A2058G substitution in the 23S rRNA gene responsible for macrolide resistance was found. DNA sequences of Ureaplasma spp./Mycoplasma spp. were determined as wild type, which was not fully consistent with the results of analysis of their antimicrobial susceptibility.

[Drug resistance mutations and susceptibility phenotypes of Neisseria gonorrhoeae isolates in Russia].

Steady growth in the degree of antimicrobial resistance in Neisseria gonorrhoeae calls for the control of the spreading of resistance mutations. Here we present the data describing drug resistance mutations, the results of antimicrobial susceptibility tests, and molecular genotypes of 128 recent N. gonorrhoeae isolates collected across 9 regions of the Russian Federation. The mutations in chromosome genes penA, ponA, rpsJ, gyrA, parC, which determine the susceptibility of N. gonorrhoeae to penicillins, tetracyclines, and fluoroquinolones were detected by multiplex amplification followed by hybridization on a hydrogel microarray. The most frequent mutation was an insertion of an aspartate at position 345 of penA gene (76.6%), whereas mutations Leu421Pro in ponA gene, Val57Met in rpsJ gene, Ser91Phe in gyrA gene, Asp95Gly in gyrA gene, and Ser87Arg in parC gene were detected in 32.8-36.7% of strains. One third of studied N. gonorrhoeae isolates harbored multiple drug resistance mutations in bacterial chromosome, resulting in the bimodal distribution of mutation profiles and related patterns of antimicrobial susceptibility. The spread of multiple resistance could be explained by the vertical transfer of the mutations resulting in the clonality of the N. gonorrhoeae population.

MOLECULAR EPIDEMIOLOGY OF TREPONEMA PALLIDUM IN BORDER REGION OF RUSSIAN FEDERATION (TUVA REPUBLIC).

The 111 strains of T. pallidum subsp. pallidum collected in Tuva Republic in 2013-2016 were typed by the arp, tpr (E, G, J) and tp0548 genes. The 7 subtypes were identified, in which the 14 d/f type was predominant (90.1%). The minor subtypes 14 b/f, 14 c/f, 14 d/g and 14 i/f constituted 0.9-1.8%. Single strains of 4 d/f H 9 d/f types (each 0.9%) previously described in China were detected in 2015. Both 9 and 14 arp gene variants were found in 3 clinical specimens for the first time in 2015-2016. Similarities in the molecular epidemiology of syphilis in Tuva Republic and Russia were demonstrated as well as differences from the T. pallidum subsp. pallidum population in China and Western Europe.

[Evaluation of the Recombinant Protein Tp0965 of Treponema Pallidum as Perspective Antigen for the Improved Serological Diagnosis of Syphilis].

UNLABELLED: BACKGRAUND. Treponemal tests based on the detection of antibodies against the Treponema pallidum antigens are the most specific methods for serological diagnosis of syphilis. Due to the inability to cultivate this bacterium in vitro, the most promising sources of antigens for diagnostics are recombinant proteins of T. pallidum. Evaluation of the analytical value of certain T. pallidum proteins is the approach to improve sensitivity, specificity, and reproducibility of syphilis serological tests, including possibilities of differential diagnosis of various forms of the disease. OBJECTIVE: The aim of the research was to evaluate the analytical values (sensitivity and specificity) of recombinant protein Tp0965 of T. pallidum as a candidate antigen for serological diagnosis of syphilis. METHODS: tp0965 gene was cloned into the expression vector pET28a and the construct was used for the transformation of E. coli BL-21 (DE3) cells and further expression and purification of the recombinant protein. The collected protein was used as T. pallidum antigen for serum analysis (ELISA) of groups of patients with various forms of syphilis (n=84) and the group of healthy donors (n = 25). RESULTS: High frequency of positive ELISA results was shown with serum of patients with syphilis, compared to the group of healthy donors. The sensitivity of serological reactions using recombinant protein Tp0965 was 98.8%, specificity--87.5%. The highest sensitivity (100%) was detected in the groups of patients with primary, secondary and early latent syphilis while in the group of patients with late latent syphilis it decreased to 95.2%. CONCLUSIONS: We concluded that due to its specificity T. pallidum recombinant protein Tp0965 can be used as a novel perspective antigen for development of syphilis serological diagnostic assays (for primary and early latent forms).

Evaluation of Oxidative Metabolism in Leukocytes during Phagocytosis of Escherichia coli Carrying Genetic Constructs soxS::lux or katG::lux.

We studied ROS generation by human peripheral blood monocytes and granulocytes during phagocytosis of Escherichia coli soxS::lux or katG::lux responding by luminescence (bioluminescence) to the development of oxidative stress. Initially high sensitivity of the bioluminescent reaction of E. coli katG::lux strain to the effects of model ROS (KO2 and H2O2) and pronounced induction of luminescence upon contact with granulocytes, whereas E. coli soxS::lux demonstrated less pronounced reaction to chemical oxidants and bioluminescence was observed primarily upon contact with monocytes. A correlation was found between quantitative characteristics of E. coli katG::lux bioluminescence and luminol-dependent chemiluminescence of leukocytes in some patients, but no dependence of this kind was noted for E. coli soxS::lux. The results can provide experimental substantiation of a new approach for evaluation of ROS production by leukocytes during phagocytosis and choosing the optimal object for these studies.

[Reactive oxygen and nitrogen species' effect on lux-biosensors based on Escherichia coli and Salmonella typhimurium].

The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H2O2 > OCl­ > NO• > RОO• > ONOO­> O 2 •- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO• > H2O2 > ONOO­ > RОO• > OCl­ > O 2 •- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS'::lux possessed a wider range of sensitivity, including H2O2 and OCl­, along with O 2 •- and NO•. Among the used reactive oxygen and nitrogen species, H2O2 showed the highest induction activity concerning to the plasmids pKatG'::lux, pSoxS'::lux and pRecA'::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison than the biosensors based on E. coli. .

[Effect of alkylresorcinols on thermal denaturation and refolding of bacterial luciferase and synthesis of heat shock proteins revealed in the luminescent molecular and cellular test systems].

Molecular and cellular luminescent biotests were used to reveal the effects of five alkylresorcinol homologues (C7-, C9-, C11-, C12-, and C18-AR) on the thermally induced denaturation and refolding ofbac- terial luciferases, as well as on the synthesis of heat shock proteins. The ARs activities were found to depend on their fine structure and concentration. Direct heat-protective effect of short-chain C7- and C9-AR on the chromatographically pure Photobactrium leiognathii luciferase/oxidoreductase was shown within broad range of concentration (10(-6)-10(-3) M). The long-chain ARs homologues exhibited a similar heat-protective effect at micromolar concentrations only, while their millimolarconcentrations have increased the sensitivity of the model proteins to thermal treatment. The recombinant strain Escherichia coli K12 MG1655 bearing constitutively expressed Vibrio fischieri luxAB genes was used to investigate theARs effect on the intracellular chaperone-independent refolding of bacterial luciferase. The functional activity of heat-inactivated enzyme was restored by micromolar concentrations of short-chain ARs, while long-chain homologues inhibited re- folding in the wide concentration range. The recombinant luminescent E. coli strain bearing the inducible ib- pA'::luxCDABE genetic construction was used to determine the effect of ARs on the synthesis of heat shock proteins (HSP). The preincubation mode of bacterial cells with long-chain alkylresorcinols led to dose-de- pendent stimulation of HSP synthesis (2.7 to 4 times) that confirmed some ARs function as "alarmones". Subsequent thermal treatment resulted in a 5-15-fold decrease of the following HSP induction compared to the control, while the number of viable cells opposite increased 1.5-4-fold. Thus, pretreatment of the bacte- rial cells with long-chain ARs resulted in their preadaptation to subsequent thermally induced stress. Short- chainARs caused less pronounced HSP suppression, although still was accompanied by increased heat resis- tance of the AR-pretreated bacterial cells.

Differential analysis of bactericidal systems of blood serum with recombinant luminescent Escherichia coli and Bacillus subtilis strains.

Luminescence intensity of recombinant Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of the natural luminescent microorganism Photobacterium leiognathi was studied under the influence of 30 individual samples of human blood serum of different component composition. A relationship was found between the level of residual bioluminescence and degree of the bactericidal effect. Moreover, the inhibition of E. coli lux+ luminescence was shown to be related to activity of the complement-lysozyme system. The reaction of B. subtilis lux+ primarily depended on the presence of ß-lysin in the blood serum. These data provide an experimental substantiation of a new method of differential analysis of humoral factors of nonspecific innate immunity with recombinant luminescent bacteria.

Fullerenolates: metallated polyhydroxylated fullerenes with potent anti-amyloid activity.

It has been revealed for the first time that sodium fullerenolate Na(4)[C(60)(OH)(∼30)] (NaFL), a water soluble polyhydroxylated [60]fullerene derivative, destroys amyloid fibrils of the Aß(1-42) peptide in the brain and prevents their formation in in vitro experiments. The cytotoxicity of NaFL was found to be negligibly low with respect to nine different culture cell lines. At the same time, NaFL showed a very low acute toxicity in vivo. The maximal tolerable dose (MTD) and LD50 for NaFL correspond to 1000 mg kg(-1) and 1800 mg kg(-1), respectively, as revealed by in vivo tests in mice using intraperitoneal drug injection. The observed pronounced anti-amyloid activity and low toxicity of NaFL make it a very promising lead drug for the development of potent fullerene-based therapeutic approaches for the treatment of amyloidoses, such as Alzheimer's disease and others.

Simultaneous evaluation of chemiluminescence and bioluminescence in a phagocytic system.

The spectra of luminol-dependent chemiluminescence of leukocytes, forming as a result of their oxygen-dependent bactericidal systems activation, and bacterial bioluminescence of Escherichia coli recombinant strain with cloned lux operon, used as the object of phagocytosis, are not identical. Mutual overlapping of these spectra reaches 87%, including overlapping of the photoemission maximums. However these spectra can be evaluated separately in the short-wave "arm" of the chemiluminescence spectrum (<420 nm) and the long-wave "arm" of the bioluminescence spectrum (>560). The kinetics of luminol-dependent chemiluminescence of phagocytes and of bacterial bioluminescence in their mixtures is characterized by mutually dependent phase-wise changes in the intensity of the analyzed parameters.

On-line determination of serum bactericidal activity using recombinant luminescent bacteria.

Intensity of luminescence quenching in recombinant strains of Escherichia coli with cloned lux-operones by human blood serum is directly proportional to the degree of bactericidal effect assessed by nephelometric and bacteriological methods. This correlation was most characteristic of E. coli with luminescence genes from Photobacterium leiognathi, which substantiates its use in the development of the kinetic bioluminescent method to determine of serum bactericidal activity. The possibility of using this method for evaluation of activity of classic and alternative pathways of compliment activation was demonstrated by using zymosan or EGTA-Mg(2+)-treated sera and C1-C5-deficient sera.

Effect of human serum on bioluminescence of natural and recombinant luminescent bacteria.

Biphasic modification of bacterial bioluminescence by human serum was revealed: bioluminescence was inhibited at high concentrations of the serum and stimulated at low concentrations. Effects of temperature and duration of exposure on bioluminescence manifested in stimulation of the inhibitory effect at higher temperature and longer exposure. The degree of inhibition of bioluminescence under in the presence of serum depends on species characteristics of the microorganism and nature of the luminescent system.