Vol 7, No 2 (2017)

We investigated immunogenic properties of proteins bound with nanoparticles. A process for producing spherical nanoparticles having size of 20 microns by polymerization of lactic acid and an optimal method of nanoparticle surface activation were described. Activated nanoparticles were used for covalent binding of model fusion protein comprising sequences of human beta-2 microglobulin and green fluorescent protein. It is shown that the nanoparticles were able to bind 3 micrograms of the protein per 1 mg of the polymer. According to the results of confocal microscopy and electrophoresis the protein is firmly adsorbed on the surface of the granules. F1 (CBA x C57BL) mice were subjected to intraperitoneal immunization with fusion protein modified nanoparticles and equivalent mixture of unmodified nanoparticles and unbound fusion protein. Blood was taken at 2 weeks after three-time intraperitoneal immunization. Antibody level to model protein was determined in mouse sera using enzyme-linked immunosorbent assay. Each of experimental and control groups comprised 39 animals. The validity of the results was evaluated using the Mann–Whitney test. It is shown that the average antibody level in the control group was 1.8 times greater than that in the experimental group. The diffe rence was significant (p < 0.004). We discuss the significance of the results in terms of development traps capable to bind virus particles in blood and to provide immune response.

Infectious mononucleosis is a widespread disease caused by certain members of Herpesviridae family. Acute infectious mononucleosis develops predominantly in children and is accompanied by an increase of the number of circulating naive CD4+ and naive CD8+ T-lymphocytes in the peripheral blood. The normalization of immunological parameters is achieved within 4–6 months after recovery and that is an indicator of a proper functioning of the immune system. CD95 and DR3 death receptors are involved in the initiation of apoptosis of naive T-lymphocytes in healthy people and in patients with infectious mononucleosis. The aim of the study was to evaluate the ability of CD95 and DR3 receptors to initiate apoptosis of naive CD4+ and naive CD8+ T-lymphocytes in children with infectious mononucleosis during convalescence. The material for the study was the samples of the peripheral blood of children who previously had infectious mononucleosis. The blood sampling was carried out again after 4–6 months after the disease. At the time of the study, children did not display clinical and laboratory signs of infectious mononucleosis. Same children who were examined earlier in the period of the development of acute infectious mononucleosis, as well as relatively healthy children were used as the comparison groups. Isolation of naive CD4+ and naive CD8+ T-lymphocytes was performed by negative magnetic immunoseparation. For specific stimulation of CD95 and DR3 receptors monoclonal antibodies were used. The level of apoptosis and expression of death receptors were evaluated by flow cytometry. Freshly isolated cells were analyzed, as well as cells cultured with the addition of appropriate monoclonal antibodies. It was shown that the recovery period was accompanied by increased apoptosis of freshly isolated naive CD4+ and naive CD8+ T-lymphocytes compared with the acute phase of infectious mononucleosis. Thus in both populations of naive T-cells showed an increase of CD95+ cells’ susceptibility to apoptosis. CD95 stimulation in the cell culture did not lead to the change in the level of apoptosis of naive CD4+ and naive CD8+ T-lymphocytes. The freshly isolated naive CD4+ and naive CD8+ T-lymphocytes DR3+ cells were resistant to apoptosis, and in the process of cultivating their sensitivity varied depending on the subpopulation belonging. Thus in the culture of naive CD4+ T-lymphocytes DR3 was not involved in the transfer of pro-apoptotic signal. In the culture of naive CD8+ T-lymphocytes DR3+ cells were possible to increase the apoptosis of DR3-negative cells. At the same time the DR3 activation by monoclonal antibodies in the culture caused the death of DR3+ naive CD8+ T-lymphocytes that naturally associated with decreased proapoptotic activity of these cells and resulted in inhibition of apoptosis of total pool of naive CD8+ T-lymphocytes. Thus, the functional ability of CD95 and DR3 receptors to trigger an apoptosis of naive T-lymphocytes in children during convalescence of infectious mononucleosis varied and depended on their belonging to naive CD4+ or naive CD8+ T-lymphocytes.

Purpose: whooping cough diagnostics by molecular-genetic methods.

Materials and methods. Under observation there were 4930 people during the period from 2012 to 2015. All samples were received in 8 schools of Moscow and the Moscow region: in 3 schools had been found children with whooping cough, in other 5 schools – only children with prolonged cough. Whooping cough diagnosis had been given earlier by bacteriological and serological methods. 430 clinical samples were studied by 2 methods: PCR with fluorescent hybridized detection of amplified products and isothermal amplification.

Results. In three of eight schools whooping cough cases at 7 children at the age of 7, 9, 11 and 15 years were revealed earlier. The diagnosis of whooping cough at them was confirmed by means of bacteriological and serological methods. 33 positive DNA samples (7.7%) are revealed. DNA-positive samples are allocated from 18 pupils and 15 employees of schools. In two schools where earlier infection sources were established, 15 DNA-positive samples from which in three cases clinical manifestations were observed are revealed. In those schools where it wasn’t earlier established a source of an infection and examinations conducted it is long the coughing children, 18 DNA-positive samples are revealed, and in two cases clinical manifestations in the form of cough were observed.

Conclusion. Performed research confirmed high efectiveness of molecular-genetic methods during pertussis infection foci examination in schools for infection source identification also amongst long coughing children. 

Species composition, susceptibility to antimicrobial agents, incidence and type of carbapenemase of carbapenemase-producing gram-negative bacteria, isolated in the Russian Research Centre of Radiology and Surgical Technologies from February 2014 to April 2016 were described. Screening of carbapenem-resistant bacteria was conducted by plating biomaterial on the chromogenic medium (“CHROMagar KPC”, DRG, France), species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the analyzer VITEK MS (bioMé- rieux, France). The sensitivity of gram-negative bacteria to antimicrobial drugs was determined on the analyzer VITEK-2 (bioMérieux), and the obtained results were interpreted in accordance with EUCAST criteria v.6.0, 2016. Genes encoding carbapenemase of KPC, OXA-48/162, VIM, IMP, NDM, OXA-51, OXA40/24, OXA-23 and OXA-58 groups were detected by multiplex real-time PCR (AmpliSens, Russia). 813 clinically relevant bacteria (602 patients) were obtained, including 405 gram-negative, among which 5.2% (21 strain from 16 patients) non-susceptible to meropenem and/or imipenem: Klebsiella pneumoniae (n = 5), Enterobacter cloacae (n = 2), Serratia marcescens (n = 1), Pseudomonas aeruginosa (n = 3), Acinetobacter baumannii (n = 10). Clinical material from 4 patients was infected by up to 3 carbapenem-non-susceptible strains of different species of gram-negative bacteria at the same time. The genes encoding acquired carbapenemases were detected in 84% of the nonsusceptible to carbapenems isolates: in A. baumannii — OXA40/24 (n = 8); in K. pneumoniae — OXA-48 (n = 1), KPC (n = 1) and NDM (n = 2); in P. aeruginosa — VIM (n = 1) and KPC (n = 1); in E. cloacae — KPC (n = 1). OXA-48 carbapenemase was also detected in one carbapenem-sensitive strain of K. pneumonia. All carbapenemase-producing strains had a phenotype of multidrug resistance. The results of the study showed that over the analyzed period, the prevalence of the carbapenem-nonsusceptible strains of gram-negative bacteria were relatively low, but all these strains were multidrug resistant and most of them possessed acquired carbapenemase genes. The species carbapenemnonsusceptible gram-negative bacteria and the types of the detected acquired carbapenemase were typical for Russia. For K. pneumoniae and P. aeruginosa no predominance of any one type of carbapenemase was revealed, while in all strains of A. baumannii the gene of OXA40/24 carbapenemase was found.