Broad range of inhibiting action of novel camphor-based compound with anti-hemagglutinin activity against influenza viruses in vitro and in vivo.

Influenza virus continues to remain one of the leading human respiratory pathogens causing significant morbidity and mortality around the globe. Due to short-term life cycle and high rate of mutations influenza virus is able to rapidly develop resistance to clinically available antivirals. This makes necessary the search and development of new drugs with different targets and mechanisms of activity. Here we report anti-influenza activity of camphor derivative 1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene-aminoethanol (camphecene). In in vitro experiments it inhibited influenza viruses A(H1, H1pdm09, H3 and H5 subtypes) and B with EC50's lying in micromolar range. Due to low cytotoxicity it resulted in high selectivity indices (74-661 depending on the virus). This effect did not depend on susceptibility or resistance of the viruses to adamantane derivatives amantadine and rimantadine. The compound appeared the most effective when added at the early stages of viral life cycle (0-2h p.i.). In direct hemagglutinin inhibition tests camphecene was shown to decrease the activity of HA's of influenza viruses A and B. The activity of camphecene was further confirmed in experiments with influenza virus-infected mice, in which, being used orally by therapeutic schedule (once a day, days 1-5 p.i.) it decreased specific mortality of animals infected with both influenza A and B viruses (highest indices of protection 66.7% and 88.9%, respectively). Taken together, these results are encouraging for further development of camphecene-based drug(s) and for exploration of camphor derivatives as highly prospective group of potential antivirals.

[Comparative study of the efficiency of methods for serodiagnosis of urogenital chlamydiasis].

The studies have established that the combined use of ChlamiScan and ImmunoComb IgG in practical health care is most effective for the laboratory diagnosis of Chlamydia infection of the urogenital tract.

[Comparative efficiency of detection of the causative agent of urogenital chlamydiasis by immunofluorescence, polymerase chain reaction, and dot immunoassay].

The diagnostic efficiency of three diagnostic kits--two commercial (for direct immunofluorescent test (DIFT) and polymerase chain reaction (PCR) and one predeveloped experimental dot-ELISA--was compared. For this, specimens (cervical or urethral scrapes) from 225 patients with suspected or verified urogenital chlamydiasis were analyzed. Dot-ELISA was shown to have a higher sensitivity and a higher specificity than both commercial kits. The coincidence of positive and negative results of dot-ELISA with DIFT and PCR was 98.6 +/- 1.4 and 100%, respectively. In addition, dot-ELISA on the "Amersham" membrane could detect cases of the disease by 7 and 1.2 times, as compared with PCR and DIFT, respectively. The prospects of the dot-ELISA kit for the rapid, simple, and cheap diagnosis of urogenital chlamydiasis are discussed.

[The mechanism of interaction between Yersinia pestis and erythrocytes, and its importance for the pathogenesis of plague].

The ability of Yersinia pestis to get inside human and murine red blood cells (RBC) was found both in vivo and in vitro experiments. Due to oxidase and catalase activities, the microorganisms induced the denaturation of hemoglobin (Hb) through RBC oxidation to H2O2 in high concentration providing the formation of haemin and transformation of haem Fe2+ into the utilizable form, Fe3+. This phenomenon was found to be common in vitro for all Y. pestis strains used in the study independently of Pgm phenotype and plasmid content, including vaccine Pgm(-) Y. pestis EV NIIEG and plasmidless Pgm(+) Y. pestis PKR-133 stains. This, probably, allows the bacteria to use Hb as an essential source of iron and porphyrins for de novo synthesis of DNA followed by effective multiplication in the mammalian organism. A correlation between the loss of the ability of RBC to transport O2 to organs and tissues and the development of progressive tissue hypoxia with specific clinical features of metHb accumulation and haemorrhagic syndrome was shown. The participation of Y. pestis phospholipases (A and D) in the destruction of RBC membranes and translocation of plague bacilli into RBC, as well as the phenomenon of polysaccharide chain lengthening depending on cultivation conditions of Y. pestis bacteria, are discussed.

[Development of an experimental immunoglobulin test system based on anti-idiotypic immunoglobulins for the determination of specific plague antibodies in serum of patients inoculated with the live plague vaccine EV NIIEG].

A new rapid, inexpensive, strictly specific, highly sensitive, and safe experimental ELISA test system based on rabbit anti-idiotypic immunoglobulins (anti-Id-ab) against Yersinia pestis lipopolysaccharide (LPS) was developed. Its efficiency was demonstrated, by using a panel of xenogenic antisera of biomodels immunized with the LPS extracted according to the Galanos procedure or the live plague vaccine EV NIIEG. In all cases, the similar proportions of positive reactions to the antigen itself or anti-Id-ab to LPS were registered and there was no significant difference in the detectable concentrations of both antigenic substances. The same data were obtained in ELISA with antisera from the human beings immunized with the vaccine irrespective of the time of vaccination or the number of injections. Whether the use of ELISA is promising in the development of a diagnostic immunoglobulin antiplaque test system is discussed.

[Extracellular resistance of Yersinia pestis to phagocytosis].

The authors were the first to perform stimulation of parasitizing condition of plague bacilli in mammalian bloodstream by addition of adequate quantity of glucose and calcium ions into the routine bacteriological medium, as well as growing the plague agent in RPMI-1640, isotonic to the serum of man and mammals susceptible to plague. Comparison of proliferative, phenotypic, and biochemical properties of fully virulent and vaccine Y. pestis strains demonstrated the advantages of RPMI-1640 medium, which provided extensive in vitro multiplication of the mentioned microorganisms similar to the bacterioemic stage of plague. Using methods of molecular microbiology and immunology, including a panel of monoclonal antibodies, the researchers demonstrated an abrupt fall of production of main plague surface species-specific antigens such as F1, 'murine' toxin/phospholipase D and fibrinolysin, followed by inhibition of biochemical activities associated with these antigenic substances in Y. pestis, as well as specific components of lipopolysaccharide. Possible molecular mechanisms of virulence and adaptive variability of plague bacteria in the extracellular conditions in mammalian organism are discussed.

[Influence of cultivation conditions on the expression of Yersinia pestis YopE].

With the use three types of nutrient media made it possible to study the specific features of the biosynthesis of YopE, one of the main effector proteins, coded by Yersinia pestis virulence plasmid. This protein was proved to be produced practically at all stages of Y. pestis parasitism in the host body. The above-mentioned antigen was found capable of being synthesized, depending on the conditions of Y. pestis cultivation, in the form of membrane-linked (extracellularly and under phagosomal conditions) or secreted substance, mainly in phagolysosome. In the latter case the maximum level of its expression was registered. The experimental confirmation of YopE localization in the form of superficially localized antigen/receptor at the period of the extracellular growth of bacteria is presented, which suggests its important role in the realization of the virulent properties of Y. pestis and, together with the known data on the protective properties of the antigen, indicates the prospects of its use as the basis for the creation of new chemical antiplague vaccine.

[Characterization of Yersinia pseudotuberculosis heat stable serovar specific polypeptides with the use of monoclonal antibodies]. / Kharakteristika termostabil'nykh serovarospetsificheskikh polipeptidov Yersiniia pseudotuberculosis s pomosh'iu monoklonal'nykh antitel.

The capacity of Y. pseudotuberculosis to express serovar specific polypeptides with different specificity of antigenic determinants was proved with the use of monoclonal antibodies (McAb). For the first time Y. pseudotuberculosis O antigens were found to have heat stable protein components carrying linear epitopes complementary to serovar specific MaAb and ensuring the serological specificity of the infective agent. The possibility of improving intraspecific classification of Y. pseudotuberculosis and their differentiation from other pathogenic Yersinia on the basis of the capacity of these bacteria for synthesizing species and serovar specific proteins is substantiated.

[Development of a competitive immunoassay based on monoclonal antibodies for the detection of specific antibodies to pseudotuberculosis pathogen]. / Razrabotka konkurentnogo immunofermentnogo analiza na osnove monoklonal'nykh antitel dlia vyiavleniia spetsificheskikh antitel k vozbuditeliu psevdotuberkuleza.

A highly sensitive, effective and rapid method--a competitive alternative to ELISA--was developed for the detection of specific antibodies to Y. pseudotuberculosis. It is based on monoclonal antibodies (MAbs) recognizing the species- or serogroup specific epitopes of Y. pseudotuberculosis. The competitive ELISA was used in testing the murine hyperimmune sera and human antisera sampled from patients with different infectious intestinal diseases including several cases of pseudotuberculosis. The use of the MAbs-based ELISA in the laboratory diagnostics of pseudotuberculosis is under discussion.

[Immunochemical characterization of Fraction I of Yersinia pestis strains by CAF1M gene]. / Immunokhimicheskaia kharakteristika fraktsii I shtammov Yersinia pestis, defektnykh po genu CAF1M.

The role of caf1M gene in biogenesis of Yersinia pestis capsule was studied in natural strains of the agent with Fra+/- phenotypes and recombinant variants with ycaA (caf1+;caf1M;caf1A+;caf1R+) locus defect. These bacteria did not form a clearly discernible capsule stained by classical methods but synthesized Cafl, whose content in the cells was many times higher than in lysates, in external cell wall, and in the medium with reference Y. pestis EV NIIEG culture (caf1+;caf1M;caf1A+;caf1R+). However Caf1 was not detected on the surface or culture fluid of natural and mutant Y. pestis cells. Exclusive role of Caf1M in Caf1 delivery to Y. pestis cell surface, but not in F1 monomer folding, was proven. Retention of lipopolysaccharide (LPS), a typical SR-LPS configuration and epitope specificity of its components was demonstrated, ensuring similar reactivity in solid-phase enzyme immunoassay with a panel of monoclonal antibodies to Y. pestis LPS. Study of immunochemical properties of antigenic substances derived from caf1M-defective Y. pestis cells by isolation of F1 showed that these substances represent an envelope protein involved in the caf1+ strains (together with Caf1) in assembly of "mature" F1 molecule as a result of posttranslation modification of various genes products. Variants of identification of Y. pestis with Fra+ phenotype by means of monoclonal antibodies to F1, fibrinolysis/coagulase, or LPS in solid-phase enzyme immunoassay are discussed.

[Monoclonal antibodies for studying antigens of protein origin in serotyping of Yersinia pseudotuberculosis]. / Monoklonal'nye antitela dlia izucheniia roli antigenov belkovoi prirody v serotipirovanii bakterii Yersinia pseudotuberculosis.

Hybridomas producing monoclonal antibodies (MAb) to Yersinia pseudotuberculosis, serovars I-IV, responsible for serovar appurtenance, were obtained. Virtually all MAbs reacted with protein antigens in immunoblotting. The only exclusion was MAb 3A2 presumably reacting with a glycoprotein epitope of complex structure. Variability of Y. pseudotuberculosis antigenic structure, depending on culturing temperature, was confirmed. Polypeptides with mono- or polydetermined antigenic specificity were determined using MAbs.

[Study of protein and carbohydrate products, synthesized by Yersinia pestis under conditions imitating the internal environment of mammalian phagolysosomal macrophages]. / Izuchenie produktov belkovoi i uglevodnoi prirody, sinteziruemykh Yersinia pestis v usloviiakh, imitiruiushchikh vnutrenniuiu sredu fagolizosom makrofagov mlekopitaiushchikh.

Acid shift (pH 4.0) of liquid nutrient medium containing 20 mM Mg2+ created conditions in vitro simulating the internal environment of phagolysosome into which Yersinia pestis captured by a macrophage get in vivo. The capacity of Y. pestis to survive and multiply under these conditions irrespective of the plasmid composition of strains was confirmed experimentally. Y. pestis possesses a specific mechanism of fibrinolytic activity inhibition, preventing proteolytic degradation under the effect of Ca-dependent polypeptide (Yops) fibrinolysin and potentiating, in addition to these latter, the production of the so-called "acid" proteins by Y. pestis, coded for by pCad2+ or chromosome, including the potentially new members of LCR family. The culturing conditions affect the length of O-specific lateral chains of Y. pestis lipopolysaccharide (LPS), which corresponds to LPS SR, but not R form.

[Synthesis of protective antigens during submerged cultivation of Vibrio cholerae]. / Dinamika sinteza protektivnykh antigenov v protsesse glubinnogo kul'tivirovaniia Vibrio cholerae.

The effectiveness of dot immunoanalysis for evaluating the dynamics of the synthesis of O-antigen, cholera toxin, neuraminidase, adhesin CFA1 in the process of the reactor cultivation of V. cholerae used for the production of oral chemical cholera vaccine is shown. The established regularities of the synthesis of the protective antigens of V. cholerae in the process of scaled-up cultivation are discussed.

[The rapid diagnosis of toxigenic Vibrio cholerae strains in dot immunological analysis with monoclonal antibodies]. / Ekspress-diagnostika toksigennykh shtammov Vibrio cholerae v dot-immunologicheskom analize s pomoshch'iu monoklonal'nykh antitel.

A method for identification of Vibrio cholerae toxigenic strains by dot immunoanalysis (DIA) with monoclonal antibodies to cholera toxin B-subunit is developed. It is based on analysis of lysates of isolated colonies of V. cholerae, grown in solid nutrient medium, broth cultures, and culture fluid. The analysis takes no more than 1-1.5 h, is highly sensitive, simple, and requires no special equipment and high qualification of specialists, which is of special importance in overall screening of humans and the environment for cholera.

[Characterization of Yersinia pestis fibrinolysin using monoclonal antibody]. / Kharakteristika fibrinolizina chumnogo mikroba s pomoshch'iu monoklonal'nykh antitel.

Immunochemical identity of Y. pestis fibrinolysin and coagulase is demonstrated using monoclonal antibodies. These substances are proven to exist as complex proteins with two independent activities. Possible causes of this phenomenon are discussed. Coagulase antigenic determinants are involved in specific fluorescence of Y. pestis cells grown at 28 degrees C. A new original method for screening the hybridomas producing monoclonal antibodies is proposed, based on inhibition of the functional activity of antigen.

[Immunoenzyme test-system for the detection of Vibrio cholerae 0139]. / Immunofermentnaia test-sistema dlia detektsii Vibrio cholerae 0139.

On the basis of antibodies to the lipopolysaccharide (LPS) and capsular antigen of V.cholerae O139 two variants of the enzyme immunoassay (EIA) system permitting the detection of the infective agent at a concentration of 1 x 10(6) microbial cells/ml were developed. Antibodies to V.cholerae O139 K-antigen and LPS were found to be highly active and specifically reacted only with homologous antigens without additional adsorption. Both variants of the EIA system, developed on the basis of antibodies to LPS or K-antigen, may be used for the detection of V.cholerae O139. The use of the latter variant making it possible to evaluate the virulence of the isolated cultures.

[Study of antigenic determinants of Yersinia pestis lipopolysaccharide using monoclonal antibodies]. / Izuchenie antigennykh determinant lipopolisakharida Yersinia pestis s pomoshch'iu monoklonal'nykh antitel.

Species- and genus-specific antigenic determinants of total serological activity of plague agent lipopolysaccharide (LPS) were detected for the first time with a panel of monoclonal antibodies (MAb) in Y. pestis core polysaccharide in the R-form. MAb specifically bind radicals on one or several monosaccharides of core LPS. Galactose, fucose, and mannose contain common antigenic determinants for enterobacteria; glucosamine, glucose, ribose, xylose, and 2-ketodeoxyoctanoic acid (KDO) contain determinants for Y. pestis and Y. pseudotuberculosis; and epitopes differentiating the two latter agents by the carbohydrate component are localized on glucosamine, mannose, xylose, and KDO. Epitopes common for the Enterobacteriaceae family and species-specific epitopes, two of which are identic to core polysaccharide radicals, are situated on Y. pestis lipid A.

[Comparative incidence of detection of specific antibodies to Yersinia pestis capsular antigen and lipopolysaccharide in humans immunized with pest vaccine]. / Sravnitel'naia chastota obnaruzheniia spetsificheskikh antitel k kapsul'nomu antigenu i lipopolisakharidu Yersinia pestis u privitykh chumnoi vaktsinoi liudei.

Two to 8 years after vaccination, specific antibodies to capsular antigen (F1) and lipopolysaccharide (LPS) are found in the blood sera of but 10 to 20% of subjects 1-4 times epicutaneously immunized with live antipest vaccine. Only regular continuous (for at least 7-15 years) antipest vaccination leads to the production of antibodies to the main antigens of Yersinia pestis, detectable in remote periods in 75.9 +/- 4.5% of vaccinees. It is noteworthy that the number of positive responses and titer of antibodies to LPS is appreciably higher than to F1. Detection of antibodies to Y. pestis is recommended for the retrospective diagnosis of pestis in both humans and animals in natural foci of this infection. Competitive enzyme immunoassay on nitrocellulose membranes with monoclonal antibodies is to be preferred for the purpose.

[An immunoenzyme test system for the identification of typical and atypical strains of the plague microbe]. / Immunofermentnaia test-sistema dlia identifikatsii tipichnykh i atipichnykh shtammov chumnogo mikroba.

The authors have developed the optimal variant of the enzyme immunoassay system that contains plague murine monoclonal and equine polyclonal immunoglobulins and identified all plague microbe strains at a high sensitivity (3.84 m.cl/ml). The cross reactivity of monoclonal antibodies with some pseudotuberculosis agent strains does not prevent from using the developed test system for laboratory diagnosis of plague, particularly while isolating atypical (capsule- or plasmid-free) variants of Y. pestis, taking into account parallel special diagnostic tests for pseudotuberculosis.

[Some immunochemical and immunobiological properties of Vibrio cholerae serovar 0139 capsule antigen]. / Nekotorye immunokhimicheskie i immunobiologicheskie svoistva kapsul'nogo antigena Vibrio cholerae 0139 serovara.

Capsular antigen (C-antigen) was isolated from culture fluid of V. cholerae serovar 0139 strain P16064 4-fold fractionation by (NH4)2SO4 after Weinblatt (1980). Electrophoretic separation in PAAG-SDS revealed two protein subunits with molecular weights 38 +/- 2 and 61 +/- 2 KD in the C-antigen and whole-cell lysates of two strains (MO45 and P16064) of V. cholerae serovar 0139. Murine polyclonal anticapsular antibodies reacted with these protein subunits in immunoblotting, as well as with two rapidly migrating carbohydrate components. After proteinase treatment of the C-antigen and cell lysates from opaque colonies these antibodies helped detect two high-molecular zones in addition to rapidly migrating ones. C-antigen protects from death 75% of mice infected with V. cholerae 0.139 and 43% (P < 0.05) of those infected with V. cholerae eltor 230. The protective properties of C-antigen are due to lipopolysaccharide (LPS) but not to the protein component. The immunogenicity of LPS contained in the C-antigen is selective, whereas LPS isolated from biomass of V. cholerae 0139 cells possesses similar protective activity towards the homologous strain (49.5%) and the agent of Eltor cholera (50.0%).