Combined immunization with attenuated live influenza vaccine and chimeric pneumococcal recombinant protein improves the outcome of virus-bacterial infection in mice.

Influenza and its bacterial complications are a leading cause of morbidity and mortality worldwide. The effect of combined immunization with live influenza vaccine and recombinant chimeric pneumococcal protein in dual infection caused by influenza H1N1 and S. pneumoniae (serotype 3) has been studied. The combined vaccine consisted of the strain A/California/2009/38 (H1N1) pdm and chimeric recombinant protein PSPF composed of immunodominant fragments of the surface virulence factors of S. pneumoniae-PsaA, PspA, and Shr1875-associated with modified salmonella flagellin. Vaccinated mice were infected with the influenza virus 24 hours before or 24 hours after the onset of pneumococcal infection. The protective effect of combined vaccination was shown on both models of viral-bacterial infection.

Influence of monostrain and multistrain probiotics on immunity, intestinal ultrastructure and microbiota in experimental dysbiosis.

The biological effects of three probiotic strains Lactobacillus rhamnosus K32, Bifidobacterium longum GT15, Enterococcus faecium L3 and their mixture were studied using a model of dysbiosis induced in rats by antibiotics. It was found that after taking different probiotics intestinal microbiota changed in a strain-specific manner. The maximal activity against pathogens was revealed after the administration of a mixture of bacterial strains under study or a single strain of enterococci. The strain E. faecium L3 showed the most activity against both Klebsiella spp. and Bacteroides fragilis. It helped to restore the original content of Faecalibacterium prausnitzii. The number of Klebsiella spp. was the same in the group receiving L. rhamnosus K32 and the group of animals, which was not consuming probiotics. Different probiotic strains included in the composition had various immunological effects. Probiotic bifidobacteria, enterococci and the mixture of three probiotics stimulated of mRNA expression of interleukin (IL)-10 in mesenteric lymph nodes. The changes in microbiota after consuming an enterococcal probiotic correlated with an increase in transforming growth factor (TGF)-ß and IL-10 content in blood serum and an increase of the intestinal mucus layer. Consumption of L. rhamnosus K32 led to the stimulation of IL-8 expression in mesenteric lymph nodes. Control group not receiving probiotics was characterised by expression of pro-inflammatory cytokines, damage of epithelial cells and the destruction of their tight junctions. The damage to the ultrastructure of the mucosa was prevented in all the groups taking probiotics.

Development of experimental GBS vaccine for mucosal immunization.

Streptococcus agalactiae, or group B streptococcus (GBS), is an important pathogen as it is the leading cause of neonatal deaths due to sepsis, meningitis or bacterial pneumonia. Although the development of an effective and safe GBS vaccine is on the agenda of many research labs, there is no GBS vaccine on the market yet. In the present study we attempted to engineer a live vaccine strain based on Bac, a surface protein of GBS, incorporated into a surface fimbrial protein of probiotic Enterococcus. The resulting strain induced specific systemic and local immune responses in mice and provided protection against GBS when administered via the intranasal, oral or intravaginal immunization routes.

[THE APPLICATION OF DOT-TECHNIQUE FOR DETECTING ANTIGENS OF ADENOVIRUS IN CLINICAL SAMPLES].

The article substantiates possibility of application of point enzyme-linked immunosorbent assay (dot-technique) for detecting viral antigens in samples from patients. To diagnose adenovirus infection conjugate of virus-specific monoclonal antibodies and peroxidase of horse-radish were used The chromatographic rectification of conjugate from free peroxidase permits diminishing background coloring of nitrocellulose membrane and therefore to increase sensitivity. The application of direct conjugates on the basis of virus-specific monoclonal antibodies increases specifcity of dot-technique and significantly shortens time period of analysis. As in case of application of direct conjugates on the basis of polyclonal serum, samples from patients require preliminary processing with detergent for preventing non-specific reactions. The dot-technique demonstrates good coincidence with data of polymerase chain reaction and after clinical trials it can be used in diagnostic of human viral infections.

Analysis of recombinant group B streptococcal protein ScaAB and evaluation of its immunogenicity.

Group B streptococcal (GBS) gene encoding the putative lipoprotein and adherence factor ScaAB was cloned and expressed in E. coli. Recombinant ScaAB protein was isolated. Signal sequence of ScaAB was found to be cleaved in the E. coli host. ScaAB recombinant protein was immunogenic in mice and antibodies against this protein were discovered in mice sera after GBS infection. The perspectives of the use of ScaAB protein in GBS vaccine are discussed.

Construction of recombinant polypeptides based on beta antigen C (Bac) protein & their usage for protection against group B streptococcal infection.

BACKGROUND & OBJECTIVES: immunocompromized adults. Approximately 50 per cent of the GBS strains carry and express the gene of BAC antigen which is capable to bind IgA. Gene encoding for the BAC antigen has been cloned and sequenced but actual IgA binding region on the protein has not been detected. The aim of the present work was to localize the region of IgA binding on Bac protein, to evaluate the role of one of the Bac protein regions MLKKIE in IgA binding, and to investigate the ability of Bac based recombinant proteins to generate protective antibodies against GBS infection. METHODS: Recombinant proteins based on beta antigen C were generated after PCR amplification of the fractions of bac gene with the following cloning of the PCR products into expression plasmids. Recombinant peptides were tested for IgA binding by immunoprecipitation and Western blot. One of the recombinant proteins expressing IgA binding was used as an antigen for immunization of mice and for GBS protection studies. RESULTS: Several bac gene constructs were generated. Their ability to bind IgA varied dramatically depending on the size of the construct and location of the fragment on the bac gene map. The smallest peptide expressing IgA binding was 14 kD in size. Amino acid substitutions in MLKKIE region facilitated IgA binding ability. Immunization of mice with recombinant Bac based peptide induced the appearance of anti-GBS antibody with high affinity level providing protection against GBS infection. INTERPRETATION & CONCLUSION: Size dependence of Bac based recombinant peptides proved that the effective IgA binding required specific folding of the protein binding IgA. Region MLKKIE could not be considered as region, responsible for IgA binding. Generation of antibodies against Bac based recombinant peptides with high titre and affinity makes these proteins a potent candidates for generating a vaccine against GBS infection.

Ionomycin and 2,5'-di(tertbutyl)-1,4,-benzohydroquinone elicit Ca2+-induced Ca2+ release from intracellular pools in Physarum polycephalum.

Calcium level in organelles of the slime mold Physarum polycephalum was monitored by chlortetracycline, a low-affinity calcium indicator. It was found that 2,5'-di(tertbutyl)-1,4,-benzohydroquinone (BHQ) at a concentration of 100 microM, but not the highly specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (1-10 microM), elicited calcium release from the CTC-stained intracellular calcium pool. Ionomycin also caused a calcium release (23.7+/-5.1%), which was less than that induced by BHQ (30.1+/-6.0%). Procaine (10 mM), a blocker of ryanodine receptor, completely abolished the responses to BHQ and ionomycin. Another blocker, ryanodine (100 microM), only slightly diminished the responses to ionomycin and BHQ. Apparently, BHQ and ionomycin acting as a Ca2+-ATPase inhibitor and an ionophore, respectively, elicit an increase in [Ca2+]i, which in turn triggers a calcium-induced calcium release (CICR) via the ryanodine receptor. Caffeine, an activator of ryanodine receptor, at a concentration of 25-50 mM produced a Ca2+-release (5.6-16.0%), which was not similar in magnitude to CICR. The response to 25 mM caffeine was only moderately inhibited by 25 mM procaine, and almost completely abolished by 50 mM procaine and 100 microM ryanodine.

Ontogeny of cholinergic and adrenergic mechanisms in the frog (Rana temporaria) heart.

Histochemical techniques, field stimulation, and application of autonomic drugs were used to study neurotransmission in the heart during ontogenesis of Rana temporaria. Cholinesterase (ChE)-containing fibers, fluorescent chromaffinlike cells, and fluorescent fibers were found first in the heart at tadpole stages 40, 40, and 50, respectively. Inhibitory cholinergic and stimulatory adrenergic responses to field stimulation first appeared at stages 39-40 and 42, respectively. Inhibitory responses to acetylcholine (ACh) and stimulatory responses to epinephrine (Epi) were observed as early as stages 31 and 32. The concentrations producing half-maximal response values for both neurotransmitters increased during development. Indirect evidence was obtained that the subsensitivity of tadpole hearts to ACh was due to increased hydrolysis of ACh by tissue ChE and that the subsensitivity of adult frog heart to Epi could be connected with a maturation of the neuronal uptake mechanism. The pA2 values for atropine and propranolol were 10 times greater in tadpoles than in adults. The main conclusion is that the cholino- and adrenoreactive systems appear in the frog heart cells before they become innervated and the sensitivity of these systems to neurotransmitters does not increase with innervation.

Partial change of the ts-phenotype of cold-adapted influenza virus strain grown in canine kidney cells in the presence of trypsin.

The cold-adapted temperature-sensitive (ts) influenza virus strain A/Leningrad/134/17/57 (H2N2) multiplied well at 32 degrees C (optimal temperature); lower titres of infectious virus were obtained in developing chick embryos at 40 degrees C. In a canine kidney (MDCK) cell line and in primary calf kidney (CK) cells an increased reproduction of the virus was found at 40 degrees C especially in the presence of trypsin. The ratios of virus titres obtained at optimal versus higher temperatures (RCT40) were by 1,000 times lower than those found in chick embryos. Polyacrylamide gel electrophoresis revealed a comparable synthesis of the cold-adapted influenza virus strain polypeptides HA, NP, M and NS in MDCK cells, regardless whether they were incubated at optimal or non-permissive temperatures.