Meningococcal, pneumococcal, streptococcal A and Haemophilus influenzae infections are manifested in different clinical forms, ranging from bacterial carriage to generalized life-threatening conditions. However, a connection between bacterial carriage and disease development has not been fully explored. A PCR assay was performed with adenoid biopsy samples collected from 112 children after planned adenotomy to detect Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, H. influenzae carriage. A DNA specific to at least one of the four studied microbial species was found in 104 samples (92.86%) so that: meningococcal DNA was detected in one sample (0.9%), pneumococcal — in 98 (87.5%), H. influenzae — in 19 (16.96%), and streptococcal A — in 42 (37.5%) samples. However, none of these species was found in 8 children (7.14%). A sole S. pneumoniae was detected in 54 samples (48.2%), whereas S. pyogenes — in 5 samples (4.5%). Moreover, two bacterial species were simultaneously as follows: N. meningitidis and S. pneumoniae — in 1 sample (0.9%), S. pneumoniae and H. influenzae — in 7 samples (6.3%); H. influenzae and S. pyogenes — in 1 sample (0.9%); S. pneumoniae and S. pyogenes — in 25 samples (22.3%). A triple combination consisting of S. pneumoniae, H. influenzae and S. pyogenes bacteria were detected together in 11 patients (9.8%). Meningococcal serogrouping revealed no connection with any of the 6 most common global serogroups responsible for epidemic incidence rise (A, B, C, W-135, X, Y). A clear tendency for prevalence of S. pyogenes DNA in adenoid pediatric biopsies in children diagnosed with “Adenoids and tonsils hypertrophy” vs. “Adenoids hypertrophy” was observed. It is noteworthy, a high relative prevalence of pneumococcal carriage (87.5%), found by us was of special importance. Pediatric carriers serving as a reservoir for virulent pneumococcal species pose a threat both for themselves and surrounding people. Thus, PCR-based data of adenoid biopsies may be a promising approach for future studies, as a potential to identify live viable but nonculturable bacteria in clinical specimens will contribute to a more accurate assessment of carriage rate of meningococci, pneumococci, H. influenzae and group A streptococci.

The purpose of study is to develop a traditional Endo's growth medium with the view of suppression of swarming of Proteus vulgaris and Proteus mirabilis. The object of study was a commercial dry Endo's growth medium. The inoculation of reference strains of enterobacteria and clinical sample (feces, urine, phlegm) was implemented according the normative documents. It is demonstrated that brining into Endo's growth medium tryptophan and sodium salts of bile acids in a particular ratio of their concentrations suppressed swarming of Proteus vulgaris and Proteus mirabilis. The obtained results demonstrated that application of enhanced Endo's growth medium significantly increases its differential and diagnostic characteristics.

The article describes immunoassay methods of determining the functional activity of the components C3 and C9 and C1 inhibitor for diagnostic and prognostic purposes in the treatment of patients. The activity of these components, as well as the activity and the amount of C1 inhibitor in the blood serum of children aged 6 months to 18 years suffering from atopic dermatitis have been analyzed before and after treatment to determine the involvement of the complement system in the pathogenesis of this disease. The developed methods of enzyme immunoassay for determination of the functional components of the complement system have shown high sensitivity and reliability. The activity of C9 component involved in the membrane attack of the complement was significantly below normal in children with atopic dermatitis. That indicates the involvement of membrane attack complex components in the skin necrotic processes. Component C3 activity is also reduced. C3 is the key component of the activation cascade involved in the inflammatory processes. Specific activity of C1 inhibitor increased before and during the treatment, indicating an increased biosynthesis of this acute phase protein. Positive tendency towards the normalization of the status of complement after treatment has been observed. Data obtained in this study indicate the involvement of complement system in the pathological process of atopic dermatitis in children.

Different modes of hypoxic exposure led to phasic changes in activities of the complement system components in rats sensitive to hypoxia starting from the first minutes of the posthypoxic period and persisting for 24 h and longer. The direction of shifts in the complement system depended on the duration and intensity of oxygen deficiency. Single one-hour interval hypoxia led to a moderate elevation of activities of virtually all the studied components. A more intense hypoxic exposure (1-h hypobaric hypoxia at a height of 5000 m) induced a biphasic response: reduction of activities of the majority of complement system components during the first hour of posthypoxic period and subsequent elevation of these activities above the normal. Exposure to severe hypobaric hypoxia (7000 m) led to a longer and more pronounced primary reduction of complement components activities, while the phase of their activity increase was blurred. Animal capacity to the formation of urgent tolerance of hypoxia was retained and increased with increasing the severity of hypoxic exposure. The complement consumption during the posthypoxic period was presumably a programmed reaction preventing hyperactivation of complement system components and essential for tolerance formation.

On the base of nucleotide sequence, coding IgA1 protease from Neisseria meningitidis serogroup B, strain ????58, which was determined from data base http://www.ncbi.nlm.nih.gov/Genbank), recombinant plasmide DNA was created comprising nucleotide sequence of IgA1 protease, strain H44/76, providing IgA1 protease expression in the host-cell (pBIGAPS1). The method of expression, isolation, purification and refolding of recombinant enzyme was developed. IgA1 protease exhibits high specificity and cleaves only IgA1, but not IgG. Immunogenic and protective properties of IgA1 protease to meningococcus of serogroup ??, ?? and ?? were shown. Obtained enzyme can be considered as perspective polyvaccine candidate for prophylaxis against meningococcus infection, induced by bacteria N. meningitidis and especially against serogroup B. References Kazeeva T.N., Shevelev A.B. / Biohimija. 2007. T.72. ??? 5. S. 603-614. Kilian M., Thomsen B., Petersen T.E., Bleeg H. / Mol.Immunol. 1983. V. 20. P. 1051–1058. Monteiro R. C., J. G. J. Van de Winkel. / Annu. Rev. Immunol. 2003. V. 21. P. 177–204 Mulks M.H., Plaut A.G. / N. Engl. J. Med. 1978. V. 299. P. 973–976 Mistry D., Stockley R.A. / Int. J. Biochem. Cell. Biol. 2006. V. 38. P. 1244–1248. Tajmurazov M.G. / Poluchenie i nekotorye svojstva meningokokkovoj IgA1-proteazy. Diss. kand. nauk. Gosudarstvennyj Nauchnyj Centr prikladnoj mikrobiologii. Obolensk, 2006. Arzese, A., & Botta, G. A. / Clinical Infectious Diseases. 1995. V. 20 (Suppl. 2): 169–171 Bachovchin WW, Plaut AG, Flentke GR, Lynch M, Kettner CA. / J Biol Chem. 1990. V. 265(7). P. 3738-3743. Plaut AG, Bachovchin WW. / Methods Enzymol. 1994. V. 244. P. 137-151. Jagudaeva E. Ju, L. S. Zhigis, O. A. Razguljaeva, V. S. Zueva, Je. Je. Mel'nikov, V. P. Zubov, L. V. Kozlov, A. M. Bichucher, O. V. Kotel'nikova, A. P. Alliluev, A. Je. Avakov, L. D. Rumsh. / Zhurn. bioorgan. himii. 2010, ??? 1. C.96-105 Alliluev A.P., Anohina I.V., Rumsh L.D., Kotel'nikova O.V., Drozhzhina E.Ju. Zhigis L.S., Jagudaeva E. Ju., Razguljaeva O.A., Zueva V.S., Kozlov L.V., Bichucher A.M., Avakov A.Je. / Vestnik RUDN. ser. Medicina. 2010. ??? 1. C. 7-12 Maniatis, T., Fritsch, E.F. and Sambrook J. / Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press. NY.1982. O.V. Kotel'nikova, O.V. Chibiskova, V.A. Nesmejanov, A.P. Alliluev, O.M. Vol'pina, D.O. Koroev, M.N. Zhmak, M.A. Titova, V.T. Ivanov / BJeBIM. 2005. ???5. C. 553-556.

On the base of nucleotide sequence, coding IgA1 protease from Neisseria meningitidis serogroup B, strain МС58, which was determined from data base http://www.ncbi.nlm.nih.gov/Genbank), recombinant plasmide DNA was created comprising nucleotide sequence of IgA1 protease, strain H44/76, providing IgA1 protease expression in the host-cell (pBIGAPS1). The method of expression, isolation, purification and refolding of recombinant enzyme was developed. IgA1 protease exhibits high specificity and cleaves only IgA1, but not IgG. Immunogenic and protective properties of IgA1 protease to meningococcus of serogroup А, В and С were shown. Obtained enzyme can be considered as perspective polyvaccine candidate for prophylaxis against meningococcus infection, induced by bacteria N. meningitidis and especially against serogroup B. References Kazeeva T.N., Shevelev A.B. / Biohimija. 2007. T.72. № 5. S. 603-614. Kilian M., Thomsen B., Petersen T.E., Bleeg H. / Mol.Immunol. 1983. V. 20. P. 1051–1058. Monteiro R. C., J. G. J. Van de Winkel. / Annu. Rev. Immunol. 2003. V. 21. P. 177–204 Mulks M.H., Plaut A.G. / N. Engl. J. Med. 1978. V. 299. P. 973–976 Mistry D., Stockley R.A. / Int. J. Biochem. Cell. Biol. 2006. V. 38. P. 1244–1248. Tajmurazov M.G. / Poluchenie i nekotorye svojstva meningokokkovoj IgA1-proteazy. Diss. kand. nauk. Gosudarstvennyj Nauchnyj Centr prikladnoj mikrobiologii. Obolensk, 2006. Arzese, A., & Botta, G. A. / Clinical Infectious Diseases. 1995. V. 20 (Suppl. 2): 169–171 Bachovchin WW, Plaut AG, Flentke GR, Lynch M, Kettner CA. / J Biol Chem. 1990. V. 265(7). P. 3738-3743. Plaut AG, Bachovchin WW. / Methods Enzymol. 1994. V. 244. P. 137-151. Jagudaeva E. Ju, L. S. Zhigis, O. A. Razguljaeva, V. S. Zueva, Je. Je. Mel'nikov, V. P. Zubov, L. V. Kozlov, A. M. Bichucher, O. V. Kotel'nikova, A. P. Alliluev, A. Je. Avakov, L. D. Rumsh. / Zhurn. bioorgan. himii. 2010, № 1. C.96-105 Alliluev A.P., Anohina I.V., Rumsh L.D., Kotel'nikova O.V., Drozhzhina E.Ju. Zhigis L.S., Jagudaeva E. Ju., Razguljaeva O.A., Zueva V.S., Kozlov L.V., Bichucher A.M., Avakov A.Je. / Vestnik RUDN. ser. Medicina. 2010. № 1. C. 7-12 Maniatis, T., Fritsch, E.F. and Sambrook J. / Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press. NY.1982. O.V. Kotel'nikova, O.V. Chibiskova, V.A. Nesmejanov, A.P. Alliluev, O.M. Vol'pina, D.O. Koroev, M.N. Zhmak, M.A. Titova, V.T. Ivanov / BJeBIM. 2005. №5. C. 553-556.

A method of the isolation and purification of IgA1 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 precipitate 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 developed. 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 µg. It was shown that IgA1 protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

The study group was comprised of 27 practically healthy children, 51 patients with acute bronchitis, 15 with chronic bronchitis and 11 with pneumonia. It was shown that changes of microbiocoenosis in back of the throat (BOT) were related to increased mucosal contamination with normal microflora and opportunistic microorganisms. The highest degree of contamination was observed in children with acute bronchitis. Normocoenosis was detected only in 13 practically healthy children. The disorders of microbiocoenosis took the form of disbiosis and acute inflammatory processes in patients with acute and chronic bronchitis and pneumonia. However, the large amount of normal flora together with the high Ig level ensured marked colonization resistance as evidenced by the values of natural colonization coefficient of nasopharyngeal epithelium (NCCNE) and balance coefficient (BC). These data suggested development of compensated secondary immunodeficiencies. In patients with acute bronchitis and pneumonia, local synthesis of Ig prevailed. It is shown that BC can be used to screen children for disorders of mucosal immunity. The presence of increased saliva IgE levels in patients with acute and chronic bronchitis supports the generally accepted concept of bronchi as a shock organ in allergic condition. It was demonstrated that IgE levels in saliva increase earlier than in serum and may be used as a prognostic criterion in patients with bronchopulmonary pathology.

IgA1-protease allocated from the culture N. meningitidis serogroup A. As original materials were used three different intermediate products of vaccine production: cultural fluid, cetavlon supernatant and cetavlon precipitate. IgA1-protease was used to evaluate their protectivity and immunogenity. It was shown, that isolated IgA1 protease from the meningococcus serogroup A is able to protect mice, infected by meningococcus serogroup B.

IgA1-protease allocated from the culture N. meningitidis serogroup A. As original materials were used three different intermediate products of vaccine production: cultural fluid, cetavlon supernatant and cetavlon precipitate. IgA1-protease was used to evaluate their protectivity and immunogenity. It was shown, that isolated IgA1 protease from the meningococcus serogroup A is able to protect mice, infected by meningococcus serogroup B.

A protease activity may be determined by means of immunoglobulins. Since proteolytic products apparently do not retain antigenic determinants of the initial substrate, the monitoring of enzymatic process may employ ELISA methods. The ELISA determination of functional activity of specific IgA1 protease has been used not only for detection of this enzyme, but also for measurement of its inhibition constants. IgG adsorbed onto a microplate was used for evaluation of total proteolytic activity. Varying pH values of the reaction medium it is possible to measure activity of neutral, alkaline and acid proteases. This approach was used for estimation total proteolytic activity of neutral proteases in blood serum. Due to high sensitivity of this method it was possible to dilute serum up to the level when serum inhibitors had not blocked enzyme activity. Assay of serum enzyme activity at acidic pH results in activation of pepsinogens and determination of pepsin activity. Measurement of a total level of serum pepsinogen activity may have diagnostic importance in gastroenterology, due to decisive contribution of pepsinogen I to the detectable activity.

Microbial spectrum and non-specific as well as specific IgA1 protease activity of isolated microorganisms were investigated in gingival liquid of patients with periodontitis. Microorganisms from the gingival liqud of these patients belonged to conditional-pathogenic obligate and facultatively anaerobic bacteria. 24 strains of microorganisms have been identified. Nonspecific proteolytic activity was found in the following microorganisms: Actinomyces israelii, Actinomyces naeslundii, Aerococcus viridans, Bifidobacterium longum, Neisseria subflave, Streptococcus parvulus, Eubacterium alactolyticum, Lactobaccilus catenoforme, Bacillus spp. Specific IgA1-protease activity and lack of proteolytic activity towards IgG was found in Streptococcus acidominimus, Streptococcus hansenii, Streptococcus salivarius, Leptotrychia buccalis, Staphylococcus haemolyticus and Neisseria sicca. No proteolytic activity was found in cultivation medium of Eubacterium alactolyticum (1 strain), Prevotella buccalis, Aerococcus viridans and Streptococcus sanguis.

A method is developed for determining the inhibition constants of substances capable of acting on the complement system at the stage of target recognition (immune complexes) by the first component of the complement (and, hence, blocking activation of the classical pathway of the complement). The ability of some drugs to inhibit the binding of subcomponent C1q to a target has been studied. It is shown that some drugs possess a pronounced ability to block the complement activation. The inhibition constants are compared to the therapeutic dozes of drugs. Some of the investigated preparations (suramin, sodium deoxiribonucleate, curcumin, heparin, sulfetron, guttalax, lysozyme) upon administration can present in the blood flow in concentrations capable of blocking the complement. The method is useful in the search for preparations capable of inhibiting the complement and in the study of side effects of medicinal preparations.

The immunoenzyme analysis and the method for the determination of IgG-containing immune complexes, carrying C1q component of the complement, were developed. In human blood sera the functional activity of components C3, complex C1r2s2, the content of C1 inhibitor and complement-activating immune complexes were determined. The comparative analysis of the activity of components C3 and C1r2s2, as well as between the content of C1 inhibitor and the activity of complex C1r2s2 for seropositive and seronegative sera, was made. Pronounced correlation for seropositive sera was observed. In addition, for seropositive sera correlation between an increase in IgG immune complexes and a drop in the functional activity of complex C1r2s2, as well as a drop in the functional activity of complex C1r2s2 and a growth in the titers of IgG antibodies to Chlamydia trachomatis, were established. The decreased functional activity of key complement components, simultaneously with the presence of complement-activating immune complexes and high titers of specific antibodies could be the diagnostic criteria of carrier state.

Methods of analysis of inhibition of complement system in vitro and in vivo have been developed for study of effects of medical drugs on the complement. The first one, ELISA method, for determination of inhibition of the first stage of complement activation includes binding of C1q subcomponent to immunoglobulin. The second method is based on capacity of mink serum to kill mice at the intravenous administration due to the action of mink complement. The effects of heparin, known anticoagulant, and suramin, used for treatment of trypanosomiasis, have been studied using these systems. The inhibition constants of binding suramin and heparin binding evaluated by the first method C1q were 411 +/- 29 micrograms/ml (or 0.287 +/- 0.020 mumole/l) and 36.4 +/- 1.7 micrograms/ml (or 2.28 +/- 0.10 mmole/l), respectively. This indicates that heparin binding with C1q in 10 times is higher, than that for suramin (as weight ratio) or 100 times higher in molar ratio. Administration of 3 mg of suramin or 0.3 mg of heparin to mice protected them against lethal action of intravenously injected 0.08 ml of mink serum. Blood concentrations of these compounds approximately correspond to inhibition constants for C1q binding, obtained using in vitro method.

For research of effect of medical product at a system of a complement the methods of analysis of inhibition of a system of a complement in vitro and in vivo are designed. The first of them is ELISA for determination of inhibition of the first stage of complement activation: binding of a subcomponent C1q with immunoglobulin. Second - supression of a lethal action of a mink complement on the mouse. As medical product - inhibitors of a complement the operating known by the inhibiting effect of heparin (known anticoagulant) and suramin (drug for treatment trypanosomiasis) is studied. By the first method was retrieved, that the constant of inhibition of binding suramin with C1q is 411 ± 29 mg/ml (or 0,287 ± 0,020 mmole/l), and the constant for heparin is 36,4 ± 1,7 mg/ml (or 2,28 ± 0,10 mmole/l), i.e. the binding of heparin with C1q in 10 times is better, than those for suramin in weight relation or in 100 times it is better - in molar. The second method is based on capacity of mink serum to kill of mice at the intravenous introducing due to action of a mink complement. Was shown, that the introducing 3 mg of suramin or 0,3 mg of heparin on the mouse cancels killing of mice called by the intravenous introducing 0,08 ml of mink serum, i.e. the efficiency of action of these inhibitors of a complement in experience in vivo also differs in 10 times, and their concentrations in a blood animal approximately correspond to constants of inhibition of C1q binding, obtained method in vitro.