Vol 10, No 4 (2020)

After exiting from the bone marrow (BM) into the circulation, mature neutrophil granulocytes undergo a set of sequential phenotypic and physiological changes collectively called “aging” in the absence of inflammation, by constitutively sensing prime signals from commensal microbiota and acquiring higher functional alertness in case of activation upon tissue damage or pathogen invasion. Physiological aging of blood neutrophils and their subsequent return to the BM result in signals modulating size and function of the hematopoietic niche. Circadian physiological infiltration of BM by neutrophils contributes to maintaining baseline level of circulating hematopoietic progenitor cells capable of regeneration and immune surveillance. Apart from the BM, neutrophils actively enter other healthy tissues, probably exerting some effects on their baseline physiologic state. Using lung tissue, it has been shown that neutrophils can “govern” action of gene set regulating cell growth, migration, proliferation, differentiation, and carcinogenesis. Neutrophils participate in destruction of endometrial tissues during desquamation phase as well as subsequent repair and physiological angiogenesis during proliferative phase of the menstrual cycle; promote wall rupture of the preovulatory ovarian follicle and oocyte exit; contribute to degradation and resorption of the corpus luteum in pregnancy failure; play an important physiological role in vascular remodeling in pregnant uterus and developing maternal immune tolerance to semi-allogeneic fetus. Neutrophils actively migrating to the surface of intestinal epithelium during local infection and/or damage stimulate epithelial restitution and recovery of its barrier function. On the other hand, neutrophils recruited into the oral cavity regulate quantitative and qualitative composition of microbial communities in oral biofilms, and ensure healthy state of periodontal structures. Being a major player and regulator in healing of skin wounds at early stage, inflammation, neutrophils not only destroy potential pathogens, but also participate in cleansing wounds from cell debris, produce cytokines, enzymes, and growth factors affecting further stages in repair process. Both apoptosis and NETosis underlying neutrophil death greatly contribute to wound healing. However, dysregulation and imbalance in both apoptosis and NETosis may lead to unfavorable consequences as well as developing chronic non-healing wounds.

Metal and metal oxide nanoparticles (NPs) are promising antibacterial agents. They have a broad antimicrobial activity against both Gram-positive and Gram-negative bacteria, viruses, and protozoans. The use of NPs reduces the possibility of the microbial resistance development. This review briefly shows the general mechanisms and the main factors of antibacterial activity of NPs. In this article, a comprehensive review of the recent researches in the field of new antimicrobial agents with superior long-term bactericidal activity and low toxicity is provided. The review gives the examples of synthesis of double and triple nanocomposites based on following oxides: CuO, ZnO, Fe₃O₄, Ag₂O, MnO₂, etc. including metal and nonmetal doped nanocomposites (for example with Ag, Ce, Cr, Mn, Nd, Co, Sn, Fe, N, F, etc.). Compared with bactericidal action of individual oxides, the nanocomposites demonstrate superior antibacterial activity and have synergistic effects. For example, the antimicrobial activity of ZnO against both Gram-positive and Gram-negative bacteria was increased by -100% by formation of triple nanocomposites ZnO—MnO₂—Cu₂O or ZnO—Ag₂O—Ag₂S. Similar effect was showed for Ce-doped ZnO and Zn-doped CuO. The present article also provides the examples of nanocomposites containing NPs and organic (chitosan, cellulose, polyvinylpyrrolidone, biopolymers, etc.) or inorganic materials with special structure (graphene oxide, TiO₂ nanotubes, silica) which demonstrate controlled release and longterm antibacterial activity. All of the considered nanocomposites and their combinations have a pronounced long-term antimicrobial effect including against antibiotic-resistant strains. They are able to prevent the formation of microbial biofilms on biotic and abiotic surfaces, have low toxicity to eukaryotic cells, demonstrate anti-inflammatory and woundhealing properties in compositions with polymers (sodium alginate, collagen, polyvinylpyrrolidone, etc.). The use of nanoscale systems can solve several important practical problems at the same time: saving of long-term antimicrobial activities while reducing the number of compounds, creation of new antimicrobial agents with low toxicity and reduced environmental impact, development of new biocidal materials, including new coatings for effective antimicrobial protection of medical devices.

High priority of soil-transmitted helminths worldwide and in the Russian Federation is due to their vast distribution and the severe pathological features they induce in humans. Recently, it was observed that awareness of clinicians regarding this disease category was markedly decreased, although no significant decline of the disease occurrence has been recorded, whereas rate of imported cases of parasitic diseases including soil-transmitted helminths like strongyloidiasis mainly originating from subtropical or tropical countries rose in non-endemic regions. Lack of alertness on diseases like strongyloidiasis impedes timely diagnostics and treatment. Global prevalence of strongyloidiasis was estimated to range within 30—100 million people, however the World Health Organization (WHO) suggests that it was underestimated as precise data in endemic countries remain unknown. The occurrence of these helminths has been recorded in regions of temperate-continental climate: Western Ukraine, Belarus, Moldova, the Caucasus, Central Asia, as well as in Eastern Europe and the Mediterranean region. In the Russian Federation locally acquired infections are frequently recorded in the Krasnodar Territory and Rostov Region. Here, based on multi-year experience in management of patients with strongyloidiasis we present our data and brief review of publications and systematic literature related to the challenges of its clinical picture, diagnostics and treatment. Life cycle, basic biological parameters of free-living helminth in nature and distinctive features of autoinfection related to strongyloidiasis were reviewed. Special attention was paid to the risk of developing severe forms (hyperinfection and disseminated strongyloidiasis) especially in immunocompromised hosts: HIV infection, radiotherapy followed by chemotherapy, long-term corticosteroid use. Difficulties in diagnosing Strongy-loides stercoralis infection are due to its polymorphic and non-specific clinical manifestations, as well as the lack of clinical knowledge and awareness about the disease. Clinical importance of parasitological methods for larvae detection was underlined. It was noted that the drug of choice in therapy of strongyloidiasis is ivermectin unapproved yet in Russia, whereas albendazole as an alternative drug exerts poorer efficacy, justifying a need to repeat treatment courses to establish full recovery from the disease.

Intestinal microbes involved in many physiological processes owner, contributes to the formation and maintenance of immune homeostasis by regulating immune responses to protect against colonization by pathogens. A special role in the differentiation of various subpopulations of T-lymphocytes play the segmental filamentous bacteria (Segmented filamentous bacteria, SFB), capable of inducing a gut-associated lymphoid tissue (GALT) differentiation proinflammatory Th17-cells and members of the genus Clostridium (cluster IV and XIVa) and Bacteroides fragilis (polysaccharide A [PSA]), stimulating the formation of regulatory T-cells (Treg) and production of suppressor of cytokine IL-10. Important metabolites of B. fragilis are short-chain fatty acids (SCFA), which are able to activate GALT cells through the FFAR2 receptor. Lowering of the SCFA concentration leads to the reduction of the number of Treg in the intestine and breaks Th17/Treg balance. These changes lead to direct reducing of mRNA FFAR2, Foxp3 expression and increasing in RORyt GALT. Therefore, the goal was to determine the level of the key in the edge immunoregulatory bacteria intestinal microflora rats and their effects on the transcriptional activity of the genes Foxp3 and RORyt in GALT with Salmonella-induced inflammation and during administration of vancomycin and B. fragilis. To determine the genus and species of bacteria, as well as their number in the microflora of rats, was used the method of polymerase chain reaction (PCR-RV) with their identification by 16S rDNA genes. To study the transcriptional activity of genes using polymerase chain reaction reverse transcription real-time (RT-PCR). During the experiment with the introduction of animals vancomycin and Salmonella there was an increase in the level of SFB and a decrease in A. muciniphila, F. prausnitzii. Also, during infecting rats with S. Enteritidis and S. Typhimurium on the background of pre-treatment with vancomycin, there was an increase in the number of SFBs against the background of a pronounced decrease in Bacteroides—Prevotela group, A. muciniphila, Clostridium spp. clusters XIV, IV, and F. prausnitzii, which led to a decrease in the expression level of Foxp3⁺ mRNA and an increase in RORyt⁺, respectively. However, administration of B. fragilis to animals receiving S. Enteritidis or S. Typhimurium against pretreatment with vancomycin caused a decrease in the level of SFB and mRNA RORyt⁺, and, conversely, increased the number of Bacteroides—Prevotela group, A. muciniphila, Clostridium spp. clusters XIV, IV, F. prausnitzii and expression of Foxp3⁺ genes, which indicates the restoration of the homeostasis of the intestinal microbiome. The obtained results showed that B. fragilis can be successfully used in the treatment of inflammatory bowel diseases or diseases with impaired intestinal barrier function.

Human papillomaviruses (HPVs) belong to highly abundant resulting in sexually transmitted virus infections, and cause cervical cancer holding place 4 among most common cancer types in women. In 2012, there were registered 266,000 death cases and 528,000 new cases. At present, three HPV prophylactic vaccines were generated worldwide: bivalent Cervarix, quadrivalent Gardasil and nonavalent Gardasil-9. Examining such vaccines uncovered that they are able to induce anti-HPV antibody production against viral antigens lacked in vaccine formula. The mechanism of such crossneutralizing antibodies recognizing antigens derived from various HPV pathogenic types remains unknown. In our study we attempted to uncover putative basis underlying cross-reactive interaction between vaccine-induced antibodies and non-vaccine antigens by bioinformatical approaches, that might allow optimize generation of future candidate vaccines and obtain more effective polyvalent immunobiological preparations against HPV. We used amino acid sequences of L1 coat protein of four top high-risk oncogenic HPV types (16, 18, 31 and 45) in the study. Work sequences were retrieved from the International Data Base of NCBI (National Center for Biotechnology Information) and aligned by using Clustal Omega’ and BioEdit software. A search and analysis of distinct antigenic determinant (epitopes) were performed by using software suite BepiPred-2.0: Sequential B-Cell Epitope Predictor, DiscoTope 2.0 Server, and SYFPEITHI. Bioinformatics data revealed pronounced potential of cross-neutralizing vaccine-induced antibodies and non-vaccines antigens derived from high-risk pathogenic types HPV 16, 18, 31 and 45 owing to the similarity in antigenic determinants (epitopes). Common linear determinants for T- and B-cells were found in all four types of L1 protein counterparts. In addition, similar three-dimensional B-cell determinants were discovered in HPV16 L1 and HPV18 L1. Antigenic determinants derived from HPV16 L1 and HPV31 L1 exhibited most close similarity. Hence, while immunizing with HPV16 L1, a more pronounced and moderate cross-reactive antibodies interacting with HPV31 L1 as well as HPV18 L1 and HPV45 L1 antigens, respectively, should be expected. Inversely, immunization with HPV18 L1might elicit active and less efficient crossneutralizing response with HPV45 L1 as well as HPV16 L1 and HPV31 L1, respectively.

In 2010—2016, blood serum samples were examined from 5539 patients, aged < 1—60 years, with clinically and laboratory confirmed measles. Primary or secondary type of immune response was determined for all measles cases. Studies were performed with children aged < 1—14 years (2381), adolescents, 15—17 years old (189), and adults aged 18—60 years (2969). Serum measles-specific IgM antibodies were measured by “VektoKor’ IgM” ELISA test system (Russia), concentration and avidity of specific IgG — by using “Anti-Measles Viruses ELISA/IgG” and “Avidity: Anti-Measles Viruses ELISA/ IgG” (Euroimmun, Germany). Primary immune response was identified based on the presence of serum measles-specific low avidity IgM and IgG antibodies, whereas secondary immune response was characterized by detecting high avidity IgM and IgG antibodies at concentration of ≥ 5.0 IU/ml. Analyzing measles-specific IgM antibodies in 2010—2016 demonstrated that measles morbidity was mainly due to children, aged 1—2 years reaching up to 39.9% of the total number of children with measles aged < 1—14 years as well as adults aged 18—40 years old comprising as high as 80.1% total number of patients aged 15—60 years. Serum measles-specific IgG testing showed that in 15.0% of cases they were detected at concentration of ≥ 5.0 IU/ml. Further serum dilution resulted in finding IgG titer ranging within 8.5—45.0 IU/ml (21.4+0.36) and high avidity antibodies in 80—100% (92.5+0.2) cases. The remaining 85.0% cases found low avidity measles-specific IgG antibodies (< 30%) at concentration of 0.2—3.46 IU/ml (1.73+0.03). An age-related analysis of our data demonstrated that all children under 14 with laboratory-confirmed measles developed primary immune response. Moreover, in 73.7% of measles patients aged 15—60 with primary immune response measles might be prevented by timely vaccination, whereas persons with “vaccine failure” comprised 26.3%. In 2010 (0.09 per 100,000 subjects) and 2016 (0.12 per 100,000 subjects), frequency of patients with “vaccine failure” during relative epidemic well-being was 35.3% and 18.2%, respectively, exceeding 9.9% (p < 0.001) serving as a hallmark 2014 high measles incidence rate (3.24 per 100,000 subjects).The data obtained indicate that measles virus circulate among people with “vaccine failure,” which may account for potential to spread and infect unprotected population cohorts as well as cause measles outbreaks during periods of epidemic well-being.

Y. pestis MLVA typing is used both to seek for similarities and differences between individual isolates upon conducting epidemiological investigations as well as for clonal clustering of intraspecies phylogenetic groups while analyzing microevolution and taxonomy issues. It cannot be ruled out that the most variable loci may be indicators allowing to approximate the unique strain-related properties circulating in certain natural plague foci. The Central Caucasian Highland Natural Plague Focus distinguished by heterogeneity of the circulating strains therein, including proline pro- and auxo-trophy, may represent a convenient model for testing this hypothesis. The purpose of our work was to assess the frequencies of the VNTR alleles associated with proline dependence among the Y. pestis strain VNTR loci, determined during previous MLVA-25 typing in the Central Caucasian Highland Natural Plague Focus. The main task was to identify the most informative sets of VNTR loci suitable for predicting proline pro— and auxotrophy (pro+, pro—). It was found that the loci ms45, ms56, ms46, ms07, ms69, ms62 displayed peak variability by allele frequencies and/or exhibited significant differences of mean allele frequencies in the pro— and pro+ strains. In particular, it was showed that the alleles of the ms45 locus contained 6 tandem repeats suggesting probability for pro+ reaching 0.944, whereas the alleles of the ms45 locus contained 7 tandem repeats with expected probability for pro— reaching 0.783. Moreover, the ms56 and ms46 contained 9 and more than 18 tandem repeats, respectively, thereby pointing at probability for pro+ equal to 0.933 and 0.818, respectively. Diagnostics for pro+/pro— phenotype by using specific statistical methods demonstrated statistical error 13.33% and 26.67% for the pro— and pro+ strains, respectively. All pro+ strains bearing a 6 tandem repeat complex from the ms45 locus, 9 tandem repeats derived from the ms56 locus and ms46 locus-derived 29—30 tandem repeats were accurately diagnosed solely based on these 3 loci. Thus, it is possible to predict some properties of Y. pestis strains based on determining the allele frequencies. While the number of MLVA typed plaque strains isolated in such natural focus has been progressively increased, it may be expected that opportunities for prognosing their properties based on determining locus tandem repeat composition having diagnostic value would be elevated.

Aim: to assess the relationship between colonization of the oral cavity with S. mutans and different genotypic characteristics and the degree of tooth decay in children.

Materials and methods. 274 children aged 5 to 17 years (153 girls and 121 boys) who received a preventive dental checkup were included in the study. The dental caries experience was assessed by the DMFT index (number of decayed, missing due to caries, and filled teeth), according to WHO recommendations. The plaque was collected with sterile wooden toothpicks from the buccal gingival margin or from fissures of the first molars and placed in 1.5 mL Eppendorf tubes, and then plated on Mitis Salivarius Agar medium (HiMedia, India). 481 strains of S. mutans were selected for further study. DNA was extracted by an express method. Amplification was performed in the CFX-96 thermal cycler (Bio-Rad, USA). Serotyping was performed by multiplex PCR. PCR products were analyzed by gel electrophoresis in 1.5% agarose gel with ethidium bromide (10 mg/mL) manufactured by Helicon, Moscow, and visualized in UV light in transilluminator UVT1 by Biokom. Genotyping was performed according to the methodology (Saarela et al., 1996) with the oligonucleotide primer OPA-02 (5’-TGCCGAGCTG-3’). Strains of S. mutans were studied for the presence of the following genes: gtfB, spaP, cnm, fruA, gtfB, htrA, comE, mutA x(I), mutA (II), mutA (III), nlmAB (IV), adcA, Smu.399, Smu.583, Smu.761, Smu.940c, Smu.1449, Smu.2130.

Results. S. mutans was isolated from all the examined children. Dental decay was detected in 82.4% of the children. Among the strains studied, all 4 serotypes were found: in children with a DMFT = 0 only serotypes k and f were detected; the predominant serotype in children with tooth decay was serotype c (74.7%). 19 genotypes of S. mutans were identified. In children without caries (DMFT = 0), S. mutans did not contain the genes spaP, comE, adcA, Smu.2130, Smu.1449, gtfB, htrA. With the increase in the DMFT index, the frequency of their detection increased. 9 genotypes of S. mutans had all 7 virulence factors. In 94.9% of children colonized by these “virulent” genotypes, high DMFT index scores were observed.

Conclusion. The data obtained indicate that only a limited number of specific strains have a cariogenic potential. Strains of S. mutans belonging to serotypes e and c with a combination of virulence genes spaP, gtfB, comE, adcA, Smu.2130, Smu.1449, and htrA were isolated from children with tooth decay. Strains without these factors did not cause any damage to the teeth. The degree of tooth decay increases with colonization by several genotypes with the combination of virulence factors described above.

The goal of our study was to examine local and serum cytokine level involved in regulating inflammation in patients with chronic endometritis combined with endometrial hyperplastic processes. On admission, all patients underwent hysteroscopy with separate diagnostic curettage followed by histological examination of samples isolated from the uterine and cervical canal mucosa. Such manipulations were indicated due to abnormal uterine bleeding as well as suspected endometrial pathology based on ultrasound examination. According to the histological examination data of the endometrial samples, all patients were divided into two groups: group I contained 45 women with CE combined with PEG without atypia; group II — 38 patients with morphologically verified CEE combined with AEG without atypia. Level of IL-1P, IL-2, IL-6, IFNy, TNFa in biological fluids (aspirate from the uterus; serum) was measured by using enzyme-linked immunosorbent assay. We found that in both groups (91.1% and 89.6%, respectively) the vast majority of patients was hospitalized due to abnormal uterine bleeding. Oligomenorrhea alternated with intermenstrual bleeding (66.7% and 71.2%, respectively) and dominated in pattern of menstrual cycle disorders in the examined patients, whereas 11 (24.4%) and 7 (18.4%) patients from group I and II, respectively, were noted to suffer from severe menstrual bleeding. Overall, analyzing the data on cytokine level both in the uterine aspirate and serum evidences about ongoing inflammatory process found at examination time point. Upon that, such process was not only local, but also exhibited signs of a systemic inflammatory response. The data on cytokine level in the uterine aspirate from patients with CE coupled to PGE or CGE without atypia point at local inflammatory process characterized by significantly increased concentration of IL-ф, IL-2, IL-6, TNFa and IFNy. At the same time, higher level of IL-ф and IFNy in patients from group II might indicates that degree of morphological changes in the endometrium could affect the level of local cytokine production. Thus, the data obtained evidence that immune changes in chronic endometritis combined with non-atypical endometrial hyperplastic processes mostly occur locally. In this regard, measuring cytokine concentration in the uterine aspirate is a diagnostic predictor and serves as a sign for monitoring therapeutic effectiveness of therapy in this cohort of patients.

Bacterial pneumonia holds the second place after respiratory tuberculosis in patients with HIV infection. In recent years, sexual transmission of HIV was replaced by injection drug route. It seems of high relevance to advance medical aid to patients with HIV infection and bacterial pneumonia depending on psychoactive substance use.

Aim of study — assessment of clinical and immunological manifestations of bacterial pneumonia coupled to HIV infection with respect to verified injection drug use.

Materials and methods. Clinical and immunological data collected from 224 patients with HIV infection and pneumonia were retrospectively analyzed: group 1 group — 70 patients with HIV infection, IDU, verified bacterial pneumonia; group 2 — 16 injecting drug users (IDU) with HIV infection and pneumonia of unverified etiology; group 3 — 65 patients with HIV and bacterial pneumonia of verified etiology without injection drug use, group 4 — 73 patients with HIV infection and bacterial pneumonia of unverified etiology, without injection drug use. The data obtained were analyzed by using software Statistica 13.3. Methods of descriptive statistics with calculation of nonparametric criterion — the Kruskall—Wallis test (H-criterion) and χ² test — were used.

Results. Immunological manifestations of HIV infection and bacterial pneumonia were characterized by decreased count of CD4⁺ cells paralleled with increased count of CD3⁺ and CD8⁺ cells at higher magnitude without injection drug use being also featured with peak viral load upon developing pneumonia. Bacterial pneumonia coupled to HIV infection showed clinical manifestations similar both in injecting drug users and non-users, proceeding in 10% cases during normothermia. Injection drug user patients often demonstrated clinical picture of pneumonia resembling those found in sepsis such as pain in the body, muscles, bone aches untypical to HIV-sexually infected subjects. In addition, systolic murmur on a heart top was more often auscultated in this patient group.

Conclusion. Subjects self-considered healthy being at risk of sexually transmitted infections should examined for HIV. All subjects manifested with symptoms of the lower airway tract infections in admission department should not be rejected to be hospitalized and undergo chest X-ray examination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the 2019 novel coronavirus (2019-nCoV) that causes acute respiratory distress syndrome (ARDS) which is the main reason for patients mortality. One of the effective treatments to reduce the effects of this virus is parthenolide (PN). Parthenolide is a sesquiterpene lactone found in medicinal plants. It can inhibit several pro-inflammatory signaling pathways, in particular the ATPase activity of NLRP3. Based on its ability to suppress inflammatory signal transduction and elevated level of serum IL-1β (a surrogate marker for NLRP3 activation) in COVID-19 patients, we suggest that PN could be potentiallyeffective for the treatment of COVID-19.

A simple accelerated method for determining «self/non-self» microorganisms by using the agglutination reaction (RA) and therapeutic/prophylactic serum (Immunoglobulin complex preparation, lyophilized IgG, IgA, IgM immunoglobulins, developed by CSC Immuno-Gem, Moscow) is proposed to test for pathogenic, opportunistic and dominant probiotic Bifidobacteria spp. In parallel, all the microbial cell cultures examined were registered in the databases of Russia-wide and international collections and tested by the intermicrobial “self/non-self” recognition method, previously developed by us. 16 collection strains of various microorganisms were assessed by the RA with relevant therapeutic and prophylactic serum. Biological samples were obtained from the collection bacterial strains of Bifidobacterium bifidum 791, Escherichia coli LEGM-18, Klebsiella pneumoniae 278, Lactobacillus fermentum 90T-C4, Bifidobacterium longum MC-42, Escherichia coli M-17, Shigella sonnei 177b, Shigella flexneri 170, Escherichia coli 157, Staphylococcus aureus 209, Candida albicans 10231 and Salmonella serovar Enteritidis ATCC 10708. In addition, cell cultures obtained from the Museum of the Institute of Cellular and Intracellular Symbiosis UB RAS such as Bifidobacterium longum ICIS-505, Lactobacillus acidophilus ICIS-1127, Bifidobacterium bifidum ICIS-202, Bifidobacterium bifidum ICIS-310 were also included into the study. To assess microbial peptidoglycan foreignness, the intermicrobial “self/non-self” recognition method was also used based on inducing metabolites produced by the “dominant” test strain Bifidobacterium longum MC-42 after pre-incubation with metabolites collected from the studied cell cultures (“associates”) followed by established “dominant-associate” feedback loop. The data were evaluated by assessing change-fold in reproduction (growth/replication) and adaptation (biofilm formation and anti-lysozyme test) of microbial cultures in accordance with the described technique followed by comparing these two methods for intermicrobial “self/non-self” recognition. All the RA data were found to fully agree with those obtained after previous studies by using intermicrobial “self/non-self” recognition method coupled to “dominant-associate” system. Moreover, compared to analogous “intermicrobial recognition” method (5 days), ease of use and test timeframe (24 hours) allow to consider RA attractive for screening studies to select strains for scientific and industrial purposes.