Search

We studied immunogenicity of two recombinant proteins FR.9 and FR.11-3 created on the basis of fragments of the primary structure of N. meningitidis IgA1 protease with different molecular weights containing different sets of T and B epitopes. The proteins actively protect animals infected with live virulent culture of meningococci, serogroups A, B, and C. Analysis of CD4 + , CD8 + , and CD19 + lymphocyte populations in mouse blood showed predominant contribution of different cell populations to the formation of immune response to different proteins. Injection of FR.11-3 protein to animals did no affect the immunoregulatory index, hence, this protein can be used for creation of immunologically safe vaccine preparation.

Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

Objectives: A new approach to estimation of IgA subclass levels and IgA1/IgA2 ratio using enzymatically active and inactive forms of Neisseria meningitidis IgA1 protease was developed., Results: The approach was tested using the sera of healthy volunteers and patients with meningococcal meningitis. There was a significant increase in the IgA1 level in patients with meningitis (mean titer 1:1546 ± 352) compared to healthy volunteers (mean titer 1:546 ± 282), while the IgA2 content remained unchanged. The IgA1/IgA2 ratio was 6.3 for the healthy volunteers and 12.8 for patients with meningitis. IgA2 for the patients with meningitis and the healthy volunteers were almost unchanged, 1:86 ± 61 and 1:121 ± 46, respectively., Conclusions: The proposed method is economical and reliable and can be used for evaluation of IgA1 and IgA2 in clinical laboratories or for research purposes.

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species - and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology.

A method of the isolation and purification of IgAl protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and sediment obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been devised. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 mug. It was shown that IgAl protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

Chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. Chitosan is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. Here we produced new water-soluble low-molecular weight chitosan (LMW-Chi) lipid derivatives and compared their ability to stimulate humoral response with the effect of unmodified LMW-Chi or its oligosaccharide derivatives. LMW-Chi effectively penetrated into macrophage-like, lymphoid and epithelial cells. It also stimulated in mice IgG production to model proteins delivered either by subcutaneous or intranasal routes. Adjuvant effect of chitosan derivatives was comparable to or lower than that of unmodified LMW-Chi. Thus, it is possible that adjuvant effect is induced by unmodified glucosamine units of chitosan.

Alpha2,3-sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35-5.65) and its catalytic fragment (70 kDa, pI 6.0-6.3) were isolated from T. cruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarine-labeled oligosaccharides with various numbers of antennas and for alpha2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.

One hundred and eighty four strains of methicillin resistant Staphylococcus aureus (MRSA) isolated in pyosurgical and burn departments of Moscow, Minsk, Omsk, Tbilisi, Vologda, Smolensk and Dushanbe were differentiated with using phages of the International Set (BIS), two collections of experimental phages and two-probe fingerprinting. More than 50 per cent of the isolates could not be typed by the BIS phages. The Experimental Collection of the N.F. Gamaleya Research Institute of Epidemiology and Microbiology (Moscow) was more useful in the differentiation in comparison to the Experimental Collection of the London Health Centre. A new approach applied by us to the establishment of our collection i.e. screening of cultures with a definite specificity of the system of the restriction-modification (by a modified phage 85) followed by their differentiation with respect to the specificity of the prophages (by the induced phages with the respective modification specificity) provided reliable and reproducible results. Our collection made it possible to classify the phage type 85 strains which as was confirmed by the fingerprinting data belonged to 3 different genotype variants. The fingerprinting had no advantages over the typing by the phages of the more extended phage set and was recommended for differentiation of the strains (14 per cent) not sensitive to the phages used. A step-by-step scheme for typing MRSA easy for use in clinical and epidemiological laboratories is described.

A new collection of phages for typing methicillin-resistant S. aureus (MRSA) is proposed. The collection includes phage 85 (modified by the MRSA strain), capable of selecting strains with the similar specificity of the restriction-modification system, and 9 MRSA-induced phages. The latter differentiate MRSA strains according to the specificity of prophages present in bacterial cells. The use of this phage collection has permitted the typing of MRSA strains insensitive to the phages of the international collection. Among these cultures an epidemic strain has been detected and the source of its spread in the burn center has been established.

The microbiological and electron-microscopic study of the transfer of conjugative (pG873) and nonconjugative (rms7 and pE994) plasmids in two systems, on nitrocellulose filters and in a fluid culture medium, was carried out. In both systems the low frequency of the transfer of plasmid pG873 or the absence of the transfer of plasmids rms7 and pE994 were observed when nonlysogenic recipients were used for crossing. The presence of prophage in recipient cells increased the rate of the detection of gentamicin-sensitive transconjugants 100-fold and provided to reveal the transfer rms7 and pE994 plasmids. The electron-microscopic study of specimens with lysogenic recipients revealed a picture which can be interpreted as the fusion of two cells. Such picture was not observed in crossings with a nonlysogenic recipient and in preparations obtained from separate donor and recipient cultures.

It was shown possible to transfer nonconjugative plasmids during joint cultivation of the donor and recipient cells by transduction and phage-mediated conjugation. In the latter case it was necessary that the phage in the medium was free and the prophage was present in the recipient cells. Differences in the regularities of the transfer of the nonconjugative plasmids mobilized by the conjugative plasmid or phage were observed.

By inducing with mitomycin C the following phages were isolated from all the tested 32 methicillin resistant strains of S. aureus: the serogroup B phage was isolated from 2 strains, the serogroup B and F phages were isolated from 5 strains and the serogroup F phage was isolated from 25 strains. The phages were divided into 5 groups by the antiphage immunity. In group 1 of the phages 4 additional phages were specified. By the specificity of the prophages in the cultures all the strains were divided into 5 groups. Group 1 of the cultures was divided into 5 subgroups (A, B, C, D and E).

Transfer of the conjugative plasmid G873 on filters and mixed cultivation of the donor and recipient cells in liquid media is described. In the both systems the use of the lysogenic recipient cells (phages of serogroups B and F) in the crossings increased mor than 100-fold the frequency of plasmid transfer. The conjugative transfer of the plasmid in the mixed cultivation system was proved. The conjugative transfer required the presence (while not obligatory) of calcium chloride and was restricted by the serum factors.

The functional activity of phagocytic cells in 52 salmonellosis patients was studied with regard to the following characteristics: percent share of phagocytosis, phagocytic index, nitro blue tetrazolium test results, digestive activity. In patients with the unfavorable course of salmonellosis (the formation of carrier state) disturbances in the bactericidal activity of neutrophils and monocytes were established. For 32 patients leukinferon was included in the complex of etiotropic and pathogenetic treatment. The preparation was introduced in 3 intramuscular injections of 10,000 I. U. at intervals of 48 hours (the course of treatment); 10 days after the last injection this course was repeated. The use of leukinferon restored the normal functioning of phagocytes and the number of T-lymphocytes.

The resistance of methicillin-resistant staphylococci to phage 85 is due to the presence of a certain system restriction modification in microbial cells. The loss of the capacity for restricting phage DNA by the cell as the consequence of the loss of the mec determinant is not accompanied by the loss of its capacity for modifying phage DNA.

Clinical trials of human leukocytic alpha-interferon for injections, leukinferon were performed in 51 patients with different forms of surgical purulent infections. It was shown that leukinferon lowered the terms of normalization of body's temperature, leukocytosis, respiratory neutrophilic outbreak and levels of active T-lymphocytes. The same was observed when leukinferon was used prophylactically in cardiosurgical patients. The effect of leukinferon depended on the level of radical operations on primary purulent foci and severity of the patient's state. Leukinferon had immunomodulatory properties and mainly influenced the system of neutrophilic phagocytes. The action was lymphocyte-mediated. The rapid effect of leukinferon makes it necessary to recommend it for treatment of patients with purulent infections as an agent of urgent immunomodulation.

The study of the staphylococcal population heterogeneity with respect to methicillin resistance by 2 methods revealed different numbers of the resistant cells in the population. Thus, when the microbial suspension was plated on an agarized medium with methicillin (50 gamma/ml), only 0.0007--0.0005 per cent of the resistant cells were found. When the colonies were replicated from a medium without methicillin to a medium containing methicillin (50 gamma/ml), 84.3--97.3 per cent of the resistant microbial cells were found in the population of the same strains. The main mechanism in the heterogeneity of the staphylococcal population with respect to methicillin resistance was impairement of the phenotype manifestation of the antibiotic resistance under definite conditions.

A method of site-specific cleavage of some tRNAs and rRNAs at the 7-methylguanine residue is described. After reduction of 7-methylguanine by sodium borohydride treatment in the presence of the exogenous carrier RNA methylated statistically by dimethylsulfate the polynucleotide chain is cut at the modified residue by aniline. It was shown that the previously used procedure which did not involve a methylated carrier RNA is not applicable for splitting rRNAs or low concentrations of tRNAs. The method described was successfully used for site-specific cleavage of 32P-terminally labelled yeast tRNAPhe and unlabelled E. coli 16S rRNA at 7-methylguanine position.

Administration of interferon to mice with staphylococcal infection promoted elimination of the bacteria from their organs, influenced the immunological indices, normalized delayed type hypersensitivity, recovered the capacity of the spleen cells to produce interferon and increased the phagocytic activity of the peritoneal macrophages against the staphylococci. Addition of alpha-interferon to antibiotic therapy of patients with purulent septic infections of staphylococcal etiology markedly increased its efficiency. Improvement of the clinical picture of the disease, normalization of the general biochemical indices of the blood and elimination of the staphylococci from the blood and inflammation foci were observed.

The antitoxic activity of leucocytic injection interferon I and immune interferon was shown in experimental erythrocyte hemolysis in the presence of staphylococcus alpha-toxin. The antitoxic effect was directly proportional to the interferon concentration in the medium and inversely proportional to the toxin concentration. Neutralization of the antiviral activity of leucocytic interferon did not lower its antitoxic effect. The highly purified and concentrated preparation of leucocytic injection interferon I and recombinant interferon had no antitoxic effect.

Administration of leukocytic interferon to patients with sepsis and purulent resorptive fever resulted in a reliable increase in the indices of the host protective factors with respect to the infections. Addition of interferon to the surgical and antibacterial treatment promoted a decrease in the periods providing the clinical effect to 19.8 days against 43.8 days in the treatment without interferon.

The influence of type I interferon on the persistence of S. typhimurium in the body of mice has been studied. The injection of the preparation of interferon has been shown to be conductive to the survival of the animals and to reduce the time of Salmonella persistence in the body. The injection of interferon enhances the phagocytic activity of macrophages in the peritoneal exudate of mice.

The dynamics of staphylococci sensitivity to antibiotics in purulent foci of 242 in patients was studied. Two criteria were used for estimation of the above process, i.e. (I) sensitivity to the antibiotics of the whole staphylococcal population isolated and (2) sensitivity to the antibiotics of separate strains isolated from the population. In 10 or more days the number of the in-patients with staphylococci resistant to streptomycin, monomycin or oxacillin in the purulent foci increased. The number of the in-patients with penicillin, neomycin or chloramphenicol resistant staphylocci was statistically doubtful. The average number of the antibiotic resistant microbial cells in the staphylococcal populations of the foci in the patients during their stay in the hospital increased 2-4 times.

After the subcutaneous injection of type I (alpha) interferon into mice their survival rate in staphylococcal infection greatly increased. At the same time duration of staphylococcal persistence in these animals and the number of persisting staphylococci were found to decrease. After the injection of interferon the splenocytes of the treated animals showed a higher capacity for interferon production. During the whole experiment the characteristics of delayed hypersensitivity in these animals showed a tendency towards normalization in comparison with those in infected mice receiving no interferon.

The authors carried out comparative study of the incidence of spontaneous plasmide-negative variants occurrence in the S. aureus 8325phi IIde population developing under various conditions in vitro and in vivo. The mean percentage of such variants in the population developing in vitro constituted 0.9 +/- 0.2 in broth, 1.2 +/- 0.3 in the exudate, 6.3 +/- 2.0 in the population developing in vivo in the subcutaneous purulent-inflammatory focus, 9.5 +/- 2.6 in the kidneys. The mean percentage of plasma-negative variants in the purulent exudate in vivo was significantly greater than in the purulent exudate in vitro. The incidence of negative variants occurrence in vivo depended on the macroorganism properties.

The concept on the epidemiological mechanism of formation of drug-resistant bacterial populations was substantiated. Investigations showed the epidemiological mechanism of formation of the drug-resistant populations of staphylococci in the purulent-inflammatory foci to be represented in 81.4 per cent of the patients by superinfection with resistant microbes. The latter group of the patients could be divided into two subgroups: in one of the subgroups (34.9 per cent) a reduction of sensitivity in the initial staphylococci was seen in the presence of donor bacteria which later either remained in the focus or were eliminated. In this case it could be supposed that there occurred a preliminary infection with the resistant staphylococci or with the transducing bacteriophages.

The effect of the incubation temperature, i.e. 37 and 30 degrees and the effect of addition of 5 per cent of sodium chloride to the nutrient medium was studied with respect to the results of sensitivity determination of staphylococci to methicillin by 3 methods, such as serial dilutions in liquid and solid media and replica method applied to separate colonies. With the use of all 3 methods incubation of the samples at a temperature of 30 degrees increased the rate of staphylococcal cultures resistant to methicillin. Addition of sodium chloride to the solid nutrient medium increased the level of detecting methicillin resistant cultures in the samples incubated at a temperature of 37 degrees and decreased it in the samples incubated at 30 degrees.

Two methods were compared for determination of drug resistant staphylococci on the nasal mucosa of patients, i. e. the routine method for determination of staphylococcal sensitivity to antibiotics and the method of direct plating out of the starting material onto agarized media containing antibiotics. Staphylococci resistant to streptomycin, tetracycline, chloramphenicol, erythromycin, monomycin, axacillin and less frequent to penicillin were found more often with the 2nd method. A method of proportions was developed for testing sensitivity of staphylococci in purulent inflammatory foci of the patients. It provided characterization of the staphylococcal population from the foci by the number of the antibiotic resistant microbial cells.

It has been established that the administration of homologous interferon to mice activates the production by splenocytes of a factor increasing the bactericidal properties of macrophages in the peritoneal exudate. The activation effect was dose-dependent. The maximum production was noted on day 3 after the injection of interferon, type I, or its inducers. Cell-producers of the factor are mainly lymphocytes.

The dynamics of sensitivity to penicillin of staphylococcal populations in purulent inflammatory foci of patients treated and not treated with antibiotics was estimated according to 4 indices. No reliable differences in the dynamics of sensitivity to penicillin were found in 2 groups of the patients, when estimation was performed with respect to the frequency of the penicillin resistant or penicillin sensitive staphylococci and detection of the penicillin resistant staphylococci by direct inoculation of the focal excretion to the medium with penicillin. A reliable increase in the percentage of the penicillin resistant staphylococci in the microbial population was observed only in the patients treated with penicillin.

Suppression of protonmotive force generators in staphylococci lead to a loss of phage infection efficiency. KCN inhibited phage infection by 49.5-53.5%; DCCD by 51.0-61.4%; CCCP by 59.2-68.8%. Suppression tock place at the stage of phage DNA transport. Valinomycin in concentration of 0.5 microM evoked dissipation of the membrane potential, nigericin caused a reduction of the gradient pH in the staphylococci membrane at a concentration of 12.0 microM. Individually, these antibiotics did not have an essential influence on the efficiency of phage infection but when combined, a maximum inhibition of phage infection (64.5%) occurred at the stage of introducing phage DNA into the bacterial cell implicating the participation of the membrane potential and pH gradient in the transportation of phage nucleic acid to the staphylococcal cell.

The frequency of the transduction of plasmids rms5, rms7, pT127, pC194, pS194 and pUB101 by phages belonging to serological group B (80, 52, 52A, 53, 85, phi 11, S2) in two systems was compared. In system 1 phages for transduction were obtained from plasmid-containing lysogenic donors in the process of induction with mitomycin C; in system 2 phages for transduction were obtained by their multiplication in plasmid-containing nonlysogenic donors. In system 1 the transduction of plasmids rms5, rms7, pT127, pS194 by phage 52A was found to occur with a greater (by 3-5 orders) frequency than in system 2 (the frequency of transduction was 10(-2) to 10(-4), and 10(-6) to 10(-8) respectively). A similar situation was observed with plasmids rms5 and rms7 and phage 52; plasmid pT127 and phage 53; but not observed with plasmids rms5 and rms7 and phages 80, phi 11 and S2; plasmids pC194 and pS194 and phage 53; plasmid pUB101 and phages 52A, 80 and phi 11; plasmids pC194, pS194 and pT127 and phage 85.

The literature and experimental data on stability of resistance ot benzylpenicillin, streptomycin, tetracycline, chloramphenicol, erythromycin, kanamycin and methicillin in staphylococci stored for prolonged periods of time on artificial nutrient media were systematized. When the staphylococcal cultures were not in contact iwth the antibiotics for 5 to 18 months, sensitive cells to separate drugs were detected in 51.0--67.4 per cent of the strains. The number of such cells in the populations of some strains ranged from 1 to 100.0 per cent. The effect of the resistance multiplicity on the stability of resistance to benzylpenicillin, tetracycline, chloramphenicol and erythromycin was not observed.

The effect of the in vivo treatment with synthetic interferon inducer B-58 on natural killer (NK) and cytostatic cell activity was studied in CBA and A/Sn mice. A marked increase in NK cell activity against target cells YAC-1 was observed in the spleen of CBA mice within 4 days after treatment. On the other hand, NK activity in A/Sn mice was not affected by B-58. However, B-58 was shown to enhance cytostatic cell activity both in CBA and A/Sn mice, when tested against target cells P 815.