A novel antivirulent compound fluorothiazinone inhibits Klebsiella pneumoniae biofilm in vitro and suppresses model pneumonia.

The problematic treatment of infections caused by multiple-resistant Klebsiella, especially in ICU, is the leading cause of prolonged hospitalization and high mortality rates. The use of antibiotics for the prevention of infections is considered unreasonable as it may contribute to the selection of resistant bacteria. In this regard, the development of drugs that will be effective in preventing infection during various invasive procedures is extremely necessary. We have shown that the developed innovative antibacterial compound fluorothiazinone (FT) that suppresses the formation of biofilms is effective in the prevention of a model pneumonia caused by a multi-resistant clinical K. pneumoniae isolate. Prophylactic use followed by treatment with FT in mice with acute pneumonia modulates the local innate immune response without suppressing protective properties in the early stages of infection, while contributing to a decrease in the bacterial load in the organs and preventing lethal pathological changes in the lungs at later stages of K. pneumoniae infection. Further development of such antivirulence drugs and their use will reduce morbidity and mortality in nosocomial infections, as well as reduce the number of antibiotics used.

Fluorothiazinon inhibits the virulence factors of uropathogenic Escherichia coli involved in the development of urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacterium associated with urinary tract infection. Due to the development of antibiotic resistance and MDR, UPEC infection has become a serious problem in the last decade. In order to combat resistance, it is necessary to develop innovative antimicrobial agents that act by different mechanisms than conventional antibiotics. Among the new therapeutic strategies, suppression of pathogen virulence has become a promising alternative, since it fundamentally reduces selective pressure and the development of resistance. In our study, we showed that the compound Fluorothiazinon suppressed UPEC's ability to form biofilms and to move using the flagellum, as well as to penetrate into cells. Prophylactic use with subsequent treatment of FT in rodent models led to an improvement in survival and significantly reduced the bacterial load in the organs of the urinary system, thereby inhibiting the development of ascending infection and preventing the development of pathological changes in prostate tissues. These results suggest that FT affects several UPEC virulence factors at once and if similar results can be found in clinical trials it can potentially be used as a new drug against UPEC.

[Single-domain adapted antibodies against Chlamydia trachomatis, preserving the development of chlamidic infection in vitro]. / Odnodomennye adaptirovannye antitela protiv Chlamydia trachomatis, podavliaiushchie razvitie khlamidiinoi infektsii v usloviiakh in vitro.

The technology for the generating of single-domain recombinant monoclonal antibodies (nanoantibodies) based on the immunization of a camel, cloning of induced sequences encoding single-domain antigen-recognizing fragments of non-canonical camel antibodies, as well as functional selection of clones of nanoantibodies by the phage display method, was used to obtain new effective tools for more efficient diagnostics of Chlamydia infection and to develop new approaches for effective therapy. Two promising nanoantibodies were obtained. They showed effective binding to extracellular and intracellular forms of C. trachomatis, and also had activity that inhibited the development of chlamydial infection in vitro.

Role of Chlamydia in Multiple Sclerosis.

Chlamydia and antibodies to them were detected by serological, molecular biological, and culture methods in the sera and cerebrospinal fluid of patients with multiple sclerosis and in the reference groups of subjects without neurological diseases. Correlations between the agent presence in the biological fluids of patients and clinical characteristics of the disease were analyzed. C. pneumoniae were more incident in the biological liquids of patients with multiple sclerosis than in healthy volunteers. On the other hand, the incidence of the agent in the patients was not high and its presence did not correlate with the clinical manifestations. C. trachomatis was equally rare in the patients and volunteers. The studies indicated the existence of a group of patients infected by C. pneumoniae in the cohort of patients with multiple sclerosis, but the impact of this agent for the disease course remains unclear.

[Neuromyelitis optica and aquaporin-associated syndromes]. / Optikomielit i akvaporin-assotsiirovannye sindromy.

Identification of aquaporin-4 antibodies in neuromyelitis optica (NMO) is highly important to provide early diagnosis for starting pathogenetic treatment, and for differential diagnosis in neuromyelitis optica spectrum disorders (NMO-SD). In this paper, we review current pathogenetic and clinical aspects of NMO and NMO NMO-SD. We present our data on the identification of aquaporin-4 antibodies in CNS disorders. Aquaporin-4 antibodies were detected in 86.36% of patients with neuromyelitis optica, in 12.5% of patients with partial syndromes and in 100% of patients with systemic lupus erythematosus with longitudinally extensive transverse myelitis or optic neuritis. There was the correlation between the extent of lesion in the spinal cord and positive aquaporin-4 antibodies.

[Chlamydia trachomatis proteasome protein as one of the significant pathogenicity factors of exciter].

Sex-related infections are a global problem. Such infections may lead to acute or chronic diseases. Chlamydia trachomatis is a dangerous and widespread pathogenicity factor that is not sensitive to conventional drugs and has no obvious symptoms. Protein CPAF is leading factor of pathogenesis. This protein inhibits the signaling pathways of host cell and supports long survival of the pathogen in the host cell. The goal of this work was to review general properties of the proteasome Chlamydia protein CPAF, its functions, and role in pathology. The role of protein CPAF in the anti-chlamydia immune reaction is discussed. The prospects of the development of promising anti-chlamydia vaccine, as well as new effective anti-chlamydia drugs are also discussed.

[Biomarkers in multiple sclerosis (a review and experimental data)].

Identification of biomarkers in multiple sclerosis is a very complex problem, and intensive work is being made in this field for the last decades. The importance of establishing biomarkers of multiple sclerosis is related to the high disease heterogeneity and lack of characteristic symptoms that leads to diagnostic, prognostic challenges and difficulties in making decisions about therapy. In this paper, we review the most important biomarkers and discuss their diagnostic and prognostic value. Experimental results on neurofilament heavy chains and antibodies to sulfatide in multiple sclerosis are presented.

[Influence of Chlamydia trachomatis type III secretion system on regulation of cytokine response].

AIM: Develop in vitro model for studying production of cytokines by monocyte cells infected with Chlamydia trachomatis mediated by type III secretion system (TTSS). MATERIALS AND METHODS: Strain C. trachomatis L2/434/Bu was used in the experiments, culture of human monocytes U-937 was infected by this strain. Level of inflammatory cytokines was measured on flow analyzer Bio-Plex 200 (Bio-Rad Laboratories). Low molecular compound LHC-342 which belongs to the class of heterocyclic compounds was used as TTSS inhibitor. RESULTS: 24 hours after the infection with C. trachomatis culture 8 analyzed cytokines are induced in U-937 cells (IL-1beta, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-gamma, TNFalpha). The most pronounced increase was observed for IL-8, GM-CSF and IFN-gamma. Introduction of TTSS inhibitor into the culture of infected cells suppressed chlamydia growth, but addition of FeSO4 restored the growth of chlamydiae. And activity associated with translocation of effector TTSS protein IncA to inclusion membrane was suppressed. Under the conditions of the obtained model of TTSS inhibition during intracellular development of C. trachomatis a significant decrease of 2 pro-inflammatory cytokines--IL-6 and IL-1beta--was observed. CONCLUSION: Cytokine response plays a key role in the protective immune response in chlamydia infection but at the same time induces immunopathologic conditions. The data obtained give reasons to assume role of C. trachomatis TTSS in the induction of this component of immune response that requires further detailed studies.

[Characteristic of early mucosal immune response in mice during intravaginal infection caused by Chlamydia muridarum].

AIM: Comparison of features of recruitment to infection focus of cells mediating early immune reactions in intravaginally infected mice that had previously received or not received covinan (progesterone analogue). MATERIALS AND METHODS: A/Sn and BALB/c line mice were used in the study. C. muridarum strain Nigg infection was carried out intravaginally or intraperitoneally. For synchronization of sexual cycle a group of mice received subcutaneously a synthetic analogue of progesterone--proligeston (covinan) at a single dose of 33 mg/kg. Acute urogenital infection was evaluated by culture method. Quantitative determination of C. muridarum DNA (including study of persistence) was carried out by real time PCR. Subpopulation structure of cell population of peritoneal and vaginal lavage was evaluated by flow cytofluorometry. RESULTS: Intravaginal infection of mice that had not received covinan resulted in a pronounced recruitment of cells into vaginal cavity at 24 hours after the infection. Influx of neutrophils, dendritic cells and T-lymphocytes was especially pronounced. Prior administration of covinan practically nullified cell recruitment to infection focus though partial preservation of subpopulations of activated dendritic cells and CD8+ T-cells was observed. CONCLUSION: In mice with artificially induced by progesterone sensitivity to chlamydias the ability of recruitment to the infection focus of cells that mediate early immune reactions is reduced, that gives evidence on the importance of these reactions for infection outcome.

[New approaches to therapy of persistent infections: elimination of intracellular Chlamydai trachomatis by exposure to low temperature argon plasma].

AIM: Study microbicidal activity of low temperature argon plasma (LTP) that is a stream of partially ionized argon having macroscopic temperature of the environment against Chlamydia trachomatis obligate intracellular parasites. Study viability of host cells in parallel. MATERIALS AND METHODS: McCoy line cells infected with C. trachomatis (Bu-434/L2 strain) were exposed to LTP obtained by using atmospheric pressure plasma SHF generator. Intracellular localization of chlamydiae was visualized by luminescent microscopy. RESULTS: Exposure of infected McCoy line cells resulted in the destruction of chlamydia inclusions and practically complete elimination of intracellular bacteria. At the same time LTP exposure did not result in immediate death of host cells, an insignificant reduction of the number of cells was observed 24 hours after the exposure to LTP. CONCLUSION: The effect of LTP for elimination of intracellular chlamydia without significant changes in viability of eukaryotic host cells was demonstrated.

[The role of the type-three secretion system of the gram-negative bacteria in regulation of chronic infections].

The role of the type-three secretion system of the gram-negative bacteria in regulation of chronic infections is discussed. Recent research showed that most of severe chronic somatic diseases are derived from chronic infection induced in the first place by infectious agents. The role of the T3SS of different species in transition from an acute infection to persistence is reviewed. Clinical and bacteriological research showed that microorganisms are persistent in the form resistant to antibiotics. That is why one of the promising targets for the development of antibacterial new-generation treatment is T3SS that conducts transport of bacteria pathogenicity factors into eukaryotic cell. The presence of this structure is necessary for the development of an acute infectious process and chronization of an infection is essential for its functioning.

Development of Chlamydial Type III Secretion System Inhibitors for Suppression of Acute and Chronic Forms of Chlamydial Infection.

The Type III secretion system (T3SS) is currently considered to be one of the main pathogenicity factors in Gram-negative bacteria, which exhibit different types of parasitizing activity. The presence of this structure is essential for the development of an acute infection; the chronicity of the infection is fundamentally dependent upon its functioning. In this regard, T3TS is one of the most promising targets for the development of broad-spectrum antimicrobial drugs that do not develop resistance and are efficacious for the acute and chronic forms of infection. The mechanism of action in drug development is based on the specific inhibition of T3SS, which should interrupt the infectious process, thereby enabling the immune system to eliminate the pathogen. As a result of pilot screening using specific cellular and bacterial tests, followed by chemical optimization and detailed characterization of the biological activity, a new class of chlamydial T3SS inhibitors was obtained. The selected compounds have obvious advantages over the currently available inhibitors of T3SS pathogens thanks to the high inhibitory activity of these compounds with minimal damaging effects on eukaryotic cells. Preclinical trials of the selected inhibitors are currently under way.

[The choice of the targets and the search for their inhibitors by means of computer simulation for the development of innovative preparations to treat of chronic bacterial infections].

Persistence is a form of interaction of pathogenic bacteria with a host aimed to promote their long-term survivalby means of inactivation of the host's protective systems via modulation of intracellular signal pathways. Persistent forms of a pathogen are refractory to traditional antibiotic therapy and cause chronic infectious diseases. Directed search for protein targets and new antibacterial drugs using computer simulation and experimental testing for the development of innovative preparations to treat chronic bacterial infections appears to have good prospects as a method for the management of persistent infections. A stepwise strategy for realization of such approach is exemplified by the search of preparations against chlamydial infection.

[Target-specific screening of antivirulence preparations for chronic infection therapy].

Global spread of clinically significant strains resistant to antibiotics necessitated the development of new approaches to generation of antibacterial preparations. Selection of virulence factors as targets for new preparations is an alternative approach to therapy of infections caused by resistant strains and chronic infectious diseases. Contemporary state of research aimed at target selection among virulence factors of pathogenic for humans bacteria that cause chronic infections, and screening of specific inhibitors of these targets are examined. Analysis of limited data of therapeutic activity of selected preparations, i.e. experimental confirmation of the proposed concept, is provided.

[Development of vaccine preparation for urogenital Chlamydiosis prophylaxis].

Chlamydia trachomatis is one of the most widespread bacterial pathogens in the world that is transmitted sexually. Thereby a development of vaccine preparation for therapy and prophylaxis of infections caused by C. trachomatis is an actual topic of scientific research across the entire world. These vaccines could be used for both prophylaxis and therapy of already established chlamydia infection and, respectively, reduce the risk of chronic condition and prevent the spread of pathogen in the population. Features of chlamydia biology that are associated with obligate intracellular parasitism and immunopathologic state induced by this agent significantly complicate developments in this field. Currently a vaccine preparation that has reached clinical trials does not exist.

[Modeling of Brucella persistence in macrophage-like cells in vitro].

AIM: To develop model of chronic brucellosis infection in macrophage-like cells in vitro and to study properties of persistence of Brucella in them. MATERIALS AND METHODS: Infection of macrophage-like cells U937 and phagocytes B10.MLM with analysis of B. melitensis 16M intracellular growth and persistence. RESULTS: Dependence of intracellular growth and persistence of B. melitensis 16M strain in macrophages U937 from infection's multiplicity (IM) and activation of U937 cells, but notfrom preliminary intracellular adaptation of Brucella was demonstrated. Main parameters of infection (IM, centrifugation during phagocytosis, and time of phagocytosis) with B. melitensis 16M strain was modeled and their influence on persistence of the strain in B10.MLM phagocytes was studied. Centrifugation during phagocytosis resulted in development of long-lasting persistence of B. melitensis 16M in B10.MLM phagocytes. Differences in persistence of Brucella in B10.MLM phagocytes compared with U937 macrophages were demonstrated. CONCLUSION: Intracellular persistence of Brucella in B10.MLM phagocytes depends from high antibacterial activity of the latter. Phagocytes B10.MLM could be used in assays for testing chemical compounds' activities against intracellular invasion and persistence of Brucella on early stages of infection.

[Target-directed search of anti-virulence drugs].

Modern medicine now encounters with problem of the absence of effective antibacterial drugs, which are able to render therapeutic effect on chronic form of infectious process. Thus, the actual objective is to develop essentially new generation of drugs, on the basis of which should lie identification of new bacterial targets playing key role in process of chronization of infection as well as selection of new physiologically active substances, which are able to render highly specific inhibitory effect on selected target. Solving of this objective is possible during realization of new approaches for search and design of new drugs and, first of all, during usage of bioinformatics methods, which enable to identify new biotargets, select most effective chemical compounds-inhibitors and optimize their pharmacological and pharmacokinetic properties. The most promising bacterial target is secretion systems of pathogenic microorganisms participating in realization of their virulent characteristics and playing major role in transition of infectious process in chronic phase. We performed synthesis of and screening for 80 compounds, which allowed to select a range of inhibitors rendering specific target-directed effect on type 3 secretion system of Chlamydia. Obtained data allow to further assess of biological and therapeutic activity of these compounds on developed models of infectious process in vivo.

[Modern aspects of diagnostics of chronic chlamydiosis caused by persisting forms of Chlamydia].

AIM: To study the possible hematogenic route of dissemination of Chlamydia trachomatis and to analyze efficacy of methods of pathogen detection in clinical specimens (sera and scraping material). MATERIALS AND METHODS: Cultural method, electron microscopy, real-time PCR, immunofluorescent assay. RESULTS: C. trachomatis was detected in blood by using 2 tests (culture and PCR) in 95.2% of patients with confirmed Chlamydia infection. Chlamydia isolated from blood had infectious properties that could point to the presence of weakly studied hematogenic route of dissemination of C. trachomatis in host's organism. Study of diagnostic value of pathogen detection in serum showed that in case of chronic diseases of urogenital tract as well as extragenital diseases, rate of C. trachomatis detection in serum was significantly higher (61.1% of cases compared to 16.7% in scraping material). CONCLUSION: It is the first time when data about possible circulation of C. trachomatis in blood of patients was obtained. Detection of C. trachomatis in serum of patients with chronic and complicated forms of chlamydiosis provides essentially new approach for direct identification of the pathogen irrespectively from localization of infection's locus.

[Effector proteins of Clamidia].

The review summarizes the recent published data on molecular mechanisms of Chlamidiae - host cell interaction, first of all on chlamydial effector proteins. Such proteins as well as III transport system proteins that transfer many effector proteins into host cytoplasm are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of the infection. Patogen proteins participating in the next stage, that is the intracellular inclusion traffic to the centrosome, are CT229 of C. trachomatis and Cpn0585 of C. pneumoniae, which interact with cellular Rab GTPases. In C. trachomatis, IncA protein plays a key role in chlamydial inclusions fusion, CT847 modulates life cycle of the host cell, LDA3 is essential in acquisition of nutrients. CPAF protease and inclusion membrane proteins IncG and CADD participate in suppression of apoptosis of infected cells. The proteases CPAF and CT441, as well as deubiquitinating ChlaDub1 protein, contribute to avoiding the immune response.

[Requirements to a medical and biologic assessment and the hygienic control of the food production received from recombinant-DNA microorganisms].

In work the characteristic of the created in the Russian Federation system of an estimation of safety of the foodstuff received from/or with use of genetically modified microorganisms (GMM) is given, at their admission to realization and the hygienic control of given production over a revolution. It is shown, that strategy of a safety at a stage of registration GMM, the established order and accepted control measures of the foodstuff received from/or with use GMM, in Russia their large-scale commercial use, and the normative-legal and methodical base based on the federal legislation on state regulation in the field of genetically engineering activity, about quality and effectively outstrip safety of foodstuff about protection of the rights of consumers, is harmonized with approaches of the international organizations.