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C.
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Editorial
Board
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1
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Contents
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2-6
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Reviews
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MECHANISMS AND REGULATION OF RNA VIRUS RECOMBINATION
Popov N. N., Kolotova T. Yu., Davidenko M.B.
This review attempts to summarize the most important data
concerning of molecular mechanisms and regulation of the viral
recombination. Two mechanisms are responsible for RNA virus
recombination, specifically replicative recombination based on
replicase template switching and nonreplicative joining among fragments
of viral origin. Retroviruses recombination occurs during DNA
synthesis, whereby reverse transcriptase undergoes template switching
between the two copackaged RNAs, However numerous questions about
molecular mechanisms of RNA recombination remain unanswered. RNA
recombination is one of the driving forces of genetic variability and
virus evolution. But significant differences in recombination frequency
were observed among various RNA viruses and retroviruses. We do not
understand conclusively why the frequency of RNA recombination varies
so much among RNA viruses but the data summarized in this review
support the hypothesis according to which not only selection forces
plays a role in the determination of the recombination rate. Numerous
virus and host factors were found to affect the rate of viral RNA
recombinants and the distribution of recombination breakpoints.
Key words: RNA viruses, retroviruses, replicative recombination,
nonreplicative recombination, template switching, recombination hotspots
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7-14
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Experimental papers |
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PREVALENCE OF POLYMORPHISM OF THE TLR 9 TYPE GENE IN PATIENTS WITH INFECTIOUS MONONUCLEOSIS CAUSED BY EPSTEIN-BARR VIRUS
Popov M.M., Liadova T.I.
Introduction. The prevalence of polymorphism -1486 T/C of TLR-9
gene in 52 patients with infectious mononucleosis (IM) caused by the
Epstein-Barr virus was studied. Based on the results obtained, three
main genotypes -1486 T/C of the gene TLR-9-TT, TC, CC, were identified.
The study of the frequency of occurrence of individual genotypes in
patients with IV revealed dominance of CC and TT genotypes in
comparison with the control group. The study of the frequency
distribution of the -1486 T/C polymorphism of the TLR-9 gene for
different genotypes showed the specificity of the changes for the CC
genotype in patients with IM and the absence of such changes for the TT
and TC genotypes. Aim of research. To establish the frequency of the
polymorphism -1486 T/C of the TLR-9 gene in patients with IM caused by
the Epstein-Barr virus. Materials and methods.
A study to determine the polymorphism -1486 T/C of the TLR-9 gene was
conducted in 52 patients with IM. Among them, women - 31 (59,6%), men -
21 (40,4%) at the age of 18 to 34 years. The control group for studying
the prevalence of the polymorphism -1486 T/C of the TLR-9 gene was 40
healthy donors. The mean age was 24,2±2,4 years, with a range from 18
to 44 years. To detect DNA VEB using the reverse transcription PCR
method with hybridization-fluorescent detection of amplification
products, Amplisens (Russia) reagent kits were used. The polymorphic
region -1486 T/ C, rs187084 of the TLR9 gene was studied by real-time
PCR amplification by determining the length of the restriction
fragments-PCR using Ncol restriction enzyme and oligonucleotide
primers. Results. An analysis
of the results of polymorphism -1486 T/C of the TLR-9 gene made it
possible to identify three main genotypes - TT, TC, CC. The allotment
frequency of the discovered -1486Т/С SNP genotypes of the gene TLR-9 in
patients with ІМ was the following: ТТ genotype – 17 % (9 patients), ТС
– 46 % (24 patients) and СС – 37 % (19 patients). In patients of the
control group wild type of TT genotype was found in 40,0% (16
patients), heterozygous ТС genotype - in 45,7% (18 patients), while
homozygous СС genotype was found in 14,3% (6 patients). An
investigation of the frequency of occurrence of individual genotypes
revealed the dominance of CC and TT genotypes in comparison with the
heterozygous genotype of the TC. The study of the frequency
distribution of the -1486 T/C polymorphism of the TLR-9 gene for
different genotypes showed the specificity of the changes for the CC
genotype in patients with IM and the absence of such changes for the TT
and TC genotypes. Analyzing the occurrence frequencies allotment of
-1486 Т/С genotypes of the gene TLR-9 in patients with ІМ statistically
significant differences of the level р<0,05 were stated for ТТ and
ТС genotypes in the group of the patients with ІМ and the control
groupі. Thus for homozygous ТТ genotype this index comprised 17% versus
40% (р<0,05), for СС genotype - 37% versus 15% (р<0,05), while
for heterozygous TC genotype the allotment of frequencies had no
statistically significant difference in comparison with the control
group indices and was found with the same frequency in the groups of
the patients under study 46% versus 45% (р>0,1). According to
the calculated index of the odds ratio presence of homozygous CC
genotype in the genome of the patients with IM is specific for the
patients with IM (CI:1,16-9,2 і OR=3,26, consequently) allowing to
estimate it as a positive association in comparison with the received
indices for homozygous TT genotype (CI:0,12-0,82 і OR=0,31,
consequently) and heterozygous ТС genotype (CI:0,46-2,4 і OR=1,05),
which are estimated as a negative TT genotype association with IM and
absence of associations with IM for TC genotype. Our study on
polymorphism -1486 TLR-9 C/C revealed a correlation with the disease of
IM, which confirms the important role of TLR-mediated signaling in the
pathogenesis of EBV infection. Investigation of polymorphism among the
receptors involved in virus recognition is necessary to determine the
genetic background associated with the risk of infection, the course of
the disease and the possible consequences of MI. This will allow to
identify risk groups among patients and to conduct timely therapy. Conclusions.
1. It was proved that in patients with IM, the polymorphism -1486 T/C
of the gene TLR-9 was detected more reliably than in the control group.
2. Distribution of frequency of occurrence of polymorphism-1486 Т/С of
gene TLR-9 allowed to reveal association of genotype СС with manifest
forms of IM.
Keywords: infectious mononucleosis, Epstein-Barr virus, Toll-like receptors, polymorphism.
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18-22
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HOMOLOGY MODELING AND MOLECULAR DYNAMICS STUDY OF MYCOBACTERIUM TUBERCULOSIS UREASE
Lisnyak Yu. V., Martynov A. V.
Introduction. M. tuberculosis urease (MTU) is an attractive target for chemotherapeutic intervention in tuberculosis by designing new safe and efficient
enzyme inhibitors. A prerequisite for designing
such inhibitors is an understanding of urease's three-dimensional (3D) structure organization. 3D structure of M. tuberculosis urease is unknown. When
experimental three-dimensional structure of a protein is not known, homology
modeling, the most commonly used computational structure prediction method, is
the technique of choice. This paper aimed to build
a 3D-structure of M. tuberculosis urease by homology modeling
and to study its stability by molecular dynamics simulations. Materials and methods. To build MTU model, five high-resolution X-ray structures of bacterial ureases with three-subunit
composition (2KAU, 5G4H, 4UBP, 4СEU, and 4EPB) have been selected as templates. For each template
five stochastic alignments were created and for each alignment, a
three-dimensional model was built. Then, each model was energy minimized and the models were ranked by quality
Z-score. The MTU model with highest quality
estimation amongst 25 potential models was selected. To further
improve structure quality the model was refined by short molecular dynamics
simulation that resulted in 20 snapshots which were rated according to their energy and the quality Z-score. The best scoring model having minimum energy was chosen as a final homology model of 3D
structure for M. tuberculosis. The final model of MTU was also validated by using PDBsum and QMEAN servers. These checks confirmed good quality of MTU
homology model. Results and
discussion. Homology model of MTU is a nonamer (homotrimer of heterotrimers, (αβγ)3) consisting of 2349 residues. In MTU
heterotrimer, sub-units α, β, and γ tightly
interact with each other at a surface of approximately 3000 Å2. Sub-unit α contains the enzyme active site with
two Ni atoms coordinated
by amino acid
residues His347, His349,
carbamylated Lys430*, His459, His485, Asp 573, Gly490. Helix-turn-helix motif (residues 524-545) forms a
mobile flap that
covers the active site and is in closed conformation impeding access to the enzyme
active site. The structural stability of MTU model was checked by molecular dynamics simulation in explicit water at 300 К
and рН 7,4. During the simulation, root mean square deviations of Сα atoms (RMSD Сα) and root mean square fluctuations (RMSF) of amino acid residues
of MTU were monitored for 60 ns. Also, the distance between the loop that covers the active site and the dinickel
center was monitored. Analysis of MD trajectory indicate that the enzyme global structure is stable and the flap covering the active center remains in closed state during the simulation time. Conclusion. Predicted three-dimensional
structure of M. tuberculosis urease can be used in the studies of
structure-function relationships of the enzyme, in designing new safe and
efficient enzyme inhibitors aimed to struggle with infectious diseases promoted
by urease activity.
Key words: Mycobacterium
tuberculosis urease, three-dimensional structure, homology modeling,
molecular dynamics simulations
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23-36
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LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE
Lisnyak Yu. V., Martynov A. V.
Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a
serious medical and social health problem. Despite intensive efforts have been
made in the past decade, there are no new efficient
anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains
of M.tuberculosis. M. tuberculosis urease (MTU), being an important factor of the bacterium viability and
virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of
urease activity. However,
the commercially available urease inhibitors are toxic and unstable, that prevent
their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a
computational drug design. The inhibitor design is significantly
easier if binding sites
on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M.
tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have
built earlier. The method places molecular probes (small organic molecules containing various functional
groups) on a dense grid
defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy
minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the
average free energy. FTSite server outputs the protein residues delineating a binding sites
and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA) and models how the dynamics of a protein would be altered in the presence
of a modulator at a specific pocket. Pockets on the enzyme are predicted using
the Fpocket algorithm. To model the reduction in
flexibility of allosteric pocket on modulator binding, the unperturbed normal modes are first calculated for the protein. The calculation is then repeated, each time perturbing one of the
pockets in the protein. These results are combined with output from Fpocket in a support
vector machine (SVM) to predict allosteric pockets on proteins. The AlloSite
server is similar to the AlloPred method in that it uses the Fpocket algorithm to
elucidate allosteric pockets, whereas AlloPred uses an approach that combines flexibility with the
Fpocket output. Results and discussion. By computational solvent mapping method FTSite, we
have explored M.tuberculosis urease nonamer surface to find sites that tend to bind small organic molecular probes representing fragments of drug molecules with diverse hydrophobic
and hydrophilic properties. The predicted three
top ranked binding sites were situated at the interfaces between chains C and
A, and chain G of neighbour trimer (and at equivalent locations in symmetrical
trimers as well). A mapping of enzymes generally
yields the most probable sites situated in a subsite of the enzyme active site.
This was not the case for MTU which active sites were inaccessible for probes
due to the closed conformation of the covering flap, and predicted binding
sites were located not far from them at the entrance into a deep pocket. To explore their possible structural and functional role, we correlated the
locations of predicted MTU binding sites and its ancillary pockets (which remain open and solvent exposed even while the flap is closed) and indicated their partial overlapping. This overlapping may suggest
that predicted sites are likely the intermediate
binding sites responsible for recruiting a ligand to another binding site deeply buried in the protein. To examine the possibility that
predicted binding sites are the sites for allostery binding we carried out the search for probable sites of
allostery binding on MTU surface by AlloPred and AlloSite servers. Predicted probable allosteric sites overlapped with binding
sites revealed by FTSite suggesting their possible function as
sites for allosteric binding. Conclusions. On the surface
of M.tuberculosis urease, there were revealed the probable ligand binding sites that appear to be the sites of allosteric binding. They may serve as promising targets for designing novel allosteric modulators as
receptor-selective anti-tuberculosis drugs.
Key words: Mycobacterium tuberculosis urease,
anti-tuberculosis drugs, allosteric binding, computational drug design.
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37-46
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SECRETORY IMMUNOGLOBULIN A AND ITS ROLE IN FORMATION
OF CLINICAL COURSE OF SHIGELLOSIS IN CHILDREN INFECTED WITH HELICOBACTER PYLORI
Kurlan N.Yu.
Introduction. Secretory immunoglobulin A (sIgA) is a crucial factor in
protection of the gastrointestinal (GI) tract mucosa directly providing the
first line of defense of the intestine from the impact of foreign antigens. At
the same time, sIgA is able to form immune complexes not only with infectious
agents and their constituent elements, which are found in the mucous membrane,
but also with those, which for some reasons overcome the epithelial barrier and
directly penetrate lamina propria. The release of sIgA from
plasma cells occurs influenced by IL-4, IL-5, IL-6, IL-10 cytokines. Formation of long-term infectious processes
as well as chronic pathology associated with increased local immunity, sIgA in
particular, is considered in a range of studies. The local immunity factors are of great importance in
combination with two or more pathogenic causative agents that can be present in
the intestine for a long time. Taking into consideration that Shigellosis
mortality rate among children is still high up to now, the issue focused on
local immunity state in children with Shigellosis, infected with Helicobacter
pylori is currently of concern. Purpose
of the study is to explore local immunity competence in children with
Shigellosis, infected with Helicobacter pylori by means of sIgA level
assessment. Materials and methods. The study involved 68 children aged from 1 to 3, who were diagnosed with Shigellosis
Sonnei of medium severity. Additionally, the determination of H. pylori in
feces and the level of sIgA in coprofiltrate were provided. Results and discussion. Significantly higher sIgA level, in
comparison with the same values of the control group, was revealed in
coprofiltrates of all children in acute period. At the same time, in children
of Group 2 sIgA content was significantly higher than the values of patients
with background infection. The qualitative sIgA
content restored and significantly did not differ from the values of the
control group in early convalescence period of Shigellosis in children without
background infection. However, significant difference was observed with acute
period value. sIgA concentration in children infected with H.
pylori in the period of clinical recovery was significantly decreased in
comparison with acute period values. It was significantly different from the
data of healthy children and the children of
Group 2. The findings obtained are indicative of imbalance of local
immunity competence of the intestine in Shigellosis in children infected with
H. pylori, especially impaired sIgA production. Insufficient sIgA secretion in
coprofiltrates of patients with Shigellosis, that has been revealed, is not
contrary to the hypothesis of some scientists concerning the capacity of
pathogenic H. pylori strains to carry out their cytotoxic effect associated
with decrease of local protective mechanisms of the GI tract mucosa as well as
system immunity, the ability to spin out of control of specific immunity
mechanisms up to development of immune-dependent inflammation forms. Taking the revealed differences of sIgA content in
coprofiltrates in Shigellosis in patients with and without background infection
with H. pylori into consideration, we have carried out the study of
correlational connection concerned with assessment of Pearson coefficient of
this value with basic clinical laboratory values. Present correlational
interactions between sIgA values in coprofiltrates of children suffering from
Shigellosis, infected with H. pylori, and frequency of development of specific
symptoms along with their duration, are indicative of sIgA role in formation of
pathogenic mechanisms of the disease course. Conclusion. Therefore, Shigellosis in children infected
with H. pylori is accompanied by substantial impairment of local immunity competence
that influences the frequency of manifestations of some clinical symptoms and
pathologic changes of laboratory values, their duration. The data obtained allow to assume as the
perspective direction in perfection of therapy of such patients use of complex
immunoglobulin medications should be aimed primarily at inflammatory processes
stopping.
Keywords. Shigellosis,
immunoglobulins, children, Helicobacter pillory
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47-50
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INFLUENCE
OF GENTAMICIN ON ENTEROCOCCI BIOFILM FORMATION
Myronenko L.G., Peretyatko O.G., Iagniuk J.A., Martynov A.V.
Introduction. Today, it is well established that almost 80% of all
infectious diseases are caused by microorganisms that exist in the form of
biofilms. Microorganisms in the biofilms acquire signs of increased resistance
to antibiotics, disinfectants and other aggressive environmental factors,
complicate the course of infectious diseases and play an important role in
their chronicity. Formation of biofilms by hospital strains of bacteria poses
a serious threat to the practical medicine. Enterococci, foremost Enterococcus faecium and Enterococcus faecalis, are the third
most common cause of hospital infections, most of which involve the use of
permanent medical equipment. Internal hospital infections gain particular importance
in intensive care units and in surgical hospitals, since the formation of
biofilms is the cause of severe catheter and fan associated infections, sepsis,
pneumonia and endocarditis. It should be noted that ineffective antibiotic
therapy of infections, accompanied by the formation of biofilms, also leads to
significant economic losses. The aim of the work was to study the effects of gentamicin and
gentamicin in combination with a penetrator on the processes of enterococci
biofilm formation. Materials
and methods. The objects of the study included 3
strains of bacteria genus Enterococcus, obtained from the bacteria museum of
the Mechnikov Institute of Microbiology and Immunology National Academy of
Medical Sciences of Ukraine: E. faecalis ATCC 29212, E. faecalis ІМІ (Х) 49 р,
E. faecium ІМІ (Х) 80. The biofilms modelling was
performed in 4-section polystyrene Petri dishes. To study the influence of
compounds on biofilm formation, a photometric method was used. The optical
density (OD) of eluates from enterococci biofilms, stained with crystal violet,
was measured with the SF-56L spectrophotometer at a wavelength of 590 nm.
Statistical processing of the obtained data was carried out by means of
nonparametric statistical methods using Microsoft Excel 2007 and STATISTICA 6.0
programs. The validity of the differences between the two related samples was
assessed by the Wilcoxon test and the Sign test. The effect of the compound was evaluated using biofilm inhibition index (BII), which was calculated according
to the formula: [(OD positive control - OD tested) / OD positive control] ×
100%. Reduction of the OD value by more than 25% in the experiment relative to
OD positive control was considered as a positive effect. Results and discussions. Analysis
of the results allowed to conclude that gentamicin at concentration of 8 mcg/ml
is capable of preventing the formation of biofilms taken in experiments with
enterococci. Further increase in the concentration of gentamicin to 64 mcg/ml
did not lead to an increase in its activity relative to biofilm formation. When
applying gentamicin at concentrations of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml and 64
mcg/ml, the index of inhibition equaled 53,8%, 56,6%, 49,9% and 49,6%
respectively. A higher inhibitory effect of gentamicin was identified for the
formation of E. faecium biofilms than for E. faecalis ones. Thus,
when applying gentamicin at concentrations of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml
and 64 mcg/ml for E. faecalis biofilm formation, the inhibition index
was equal to 45,5 %, 46,7 %, 49,6 % and 48,5 %, for E. faecium – 54,8 %,
53,5 %, 65,3 % and 60,2 % respectively. One
way of facilitating the transportation of biocides through the extracellular
matrix of biofilms to the target of action may be the use of so-called
penetrators. Polyethylene glycol was used as a penetrator (PNT) in our studies.
Inhibition index analysis showed a statistically significant increase in
the suppressing effect of the combination of gentamicin and PNT on the
enterococci biofilm formation compared to the effect of gentamicin without PNT
(р<0,05). The inhibition index of gentamicin at concentartions of 8 mcg/ml,
16 mcg/ml, 32 mcg/ml and 64 mcg/ml with PNT amounted to 73,5 %, 74,4 %,
77,2 % and 78,2 % respectively. Also, a higher suppressing effect of gentamicin
with penetrator on enterococci biofilm formation was found for E. faecium
compared to E. faecalis. Thus, the results obtained suggest that
polyethylene glycol can increase the penetration of gentamicin through the
biofilm glycocalyx. Conclusion. Gentamicin at
concentrations of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml and 64 mcg/ml shows an
inhibitory effect on enterococci biofilm formation (inhibition index varied
from 49,6 % to 56,6 %). Inhibitory effect of gentamicin at
concentrations of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml and 64 mcg/ml on enterococci
biofilm formation is enhanced under the influence of polyethylene glycol
(inhibition index – from 73,5 % to 78,2 %).
Keywords. Gentamycin, Enterococcus, biofilm formation,
Polyethylene Glycol -400
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51-55
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INTEGRAL EVALUATION OF THE
CYTOKINE SYSTEM IN VIRAL MYOCARDITIS
Peremot S. D., Volyansky A. Y., Smelyanskaya M. V., Kashpur N.
V., Yudin I.P., Klysa T.L.
The need for an individual approach in the choice of
means for the prevention of complications in inflammatory processes in
cardiomyocytes, the course of which unfolds against a persistent viral
infection, dictates the need to determine the general mechanisms for
maintaining and progressing of the pathological process and an objective
evaluation of immunological changes. The
aim of the study was to determine changes in the system of inflammatory
mediators in patients with subacute and chronic herpesviral infectious
myocarditis on the basis of an integral assessment of the levels of opposing
groups of cytokines. Materials & methods. To achieve this goal, we conducted a
determination and analysis of changes in the cytokine profile in 87 patients
with subacute (from 2 to 6 months) and chronic (more than 6 months) myocarditis
due to an integral assessment of the mediator levels of inflammation of
opposing groups in patients with herpesviral myocarditis on treatment in
medical institutions of the Kharkov city. The average age of the patients was
(27 ± 7.4) years. The control group was attracted to 40 people without clinical
manifestations of cardiovascular diseases and in whose anamnesis there were no
data on the transferred inflammatory diseases of the myocardium. Both groups of
subjects were comparable in age and gender. The main group of subjects was
divided into two subgroups. The first was 44 patients with subacute flow, the
second - 43 patients with chronic infectious myocarditis. The diagnosis was
established in accordance with the recommendations of the Association of
Cardiologists of Ukraine and experts of the European Society of Cardiology,
according to the formation of definitions of diseases in the International
Classification of Diseases (ICD-10) of the tenth revision. The removal of
material from patients was carried out according to the rules for the collection
of infectious material. The concentration of cytokines: IL-2, IL-4, IL-6,
IL-10, INF-γ, TNF-α in serum was measured by enzyme-linked immunosorbent assay
using commercial enzyme immunoassay kits for Thermo Scientific ™ (IL-2R IL-4,
IL-6, IL-10, TNF alpha, IFN gamma ELISA Kit, Human, USA) and Stat Fax 303 Plus
spectrophotometer. Statistical processing of all received data was carried out
on a personal computer using the program Statistica, version 6.1 (StatSoft
Inc., USA) [1]. Results &
discussion. Analysis of levels of pro- and anti-inflammatory cytokines
in patients indicates an imbalance in their system, which is characterized
primarily by a significant increase in the level of IL-6 prophylaxis to (134.09
± 22.72) pg / ml (control level 11.83 ± 1, 64 pg / ml) and a relatively
moderate increase in IL-2 and TNF-α levels in subacute myocarditis. Such an
increase in the level of IL-6, in our opinion, is due to the dualism of the
action of this interleukin, the proinflammatory nature of its action at the
final stage of inflammation changes to anti-inflammatory. As a consequence, in
combination with IL-10, it limits the secretion of TNF-α. That is why its level remains high and with
chronic herpesviral myocarditis and exceeds the level of the control group more
than 8 times. In addition, in the chronic form of the course of herpesviral
myocarditis, an increase in the levels of anti-inflammatory IL4 and IL-10
cytokines is observed in 2.9 and 3.1 times, respectively. And the level of
IL-10 increased not only in comparison with the level of the control group, but
also exceeded by 1.5 times the corresponding index for subacute myocarditis. In
order to optimize the analysis of cytokine imbalance, an integral assessment of
the levels of inflammatory mediators from opposing groups was carried out.
Calculation of the integral indicator (II) of the cytokine balance was
performed by determining the values of cytokine indices as the ratios of the
levels of proinflammatory and anti-inflammatory sera in the examined patients
to the reference values of the control group and the arithmetic mean for each
opposing group of cytokines expressed in conventional units (c.u.). The optimal
balance of cytokines corresponded to the level of II ≤ 1 c.u. and indicated the
absence of inflammation, but the activity of the inflammatory process was
characterized by exceeding the level of more than 1 condition. In the group of
patients with subacute myocarditis, II was 6,27 c.u., exceeding the
corresponding calculated indicator of a group of patients with chronic course
in more than 1.6 times (3,82 c.u.). Therefore, the higher the deviation of the
II from 1 cu is, the deeper the violation of immunological homeostasis. Conclusion.
It was found that imbalance
in the cytokine system in subacute and chronic herpesviral myocarditis is a
universal immune system response, which is characterized by an increase in the
levels of proinflammatory cytokines against a background of moderate growth of
anti-inflammatory ones. The level of the integral cytokine index is more than 1
cu indicates the dysfunction of the immunological status of the patients being
examined and can be used as an additional diagnostic criterion for the
unfavorable course of the disease with a propensity to progress. Calculation of
II defines a personalized diagnosis of cytokine imbalance with the ability to
determine on its basis therapeutic approaches and the choice of
immunorehabilitation tools, and also allows evaluating the effectiveness of
selected anti-inflammatory agents for treatment of infectious herpesvirus
myocarditis.
Keywords. Cytokine, viral
myocarditis, herpesviruses.
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56-61
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NEW FORMYL PEPTIDE HAS THE STIMULATING PROPERTIES IN
POINT OF IMMUNE SYSTEM CELLS
Martynov A. V., Romanova
E. A., Pohorila M. S., Shcherbak O. M., Sidorenko T. A.,
Igumnova N. I., Yukhimenko V. I.
Introduction Tuberculosis infection (TBI) is still one of the
major health problems, despite of global intensive medical and pharmaceutical
efforts as it is known, in the majority of immunocompetent individuals TBI is
repressed by immune system, and as a result we can observe the latent TBI. The
main danger hides in unpredictable activation process of latent TBI determining
the spreading of infection among population. Now we investigate the ability of
new formyl peptide to stimulate phagocytosis completion in vivo. This strategy
is explained by the key role of the phagocytosis completion in preventing
long-term persistence of M. tuberculosis
in macrophage. Formyl peptides are released by microbes and damaged tissues
that are perceived as danger signals and is recognized by the innate immune
system by formyl peptides receptors expressed on neutrophil granulocytes.
Recent studies show that activated formyl peptide receptors (FPR1 and FPR2)
trigger a variety of functions, including chemotaxis, degranulation, ROS (reactive
oxygen) production and phagocytosis.
Materials and methods The ability of new formyl peptide to activate the
completeness of phagocytosis by peritoneal macrophages absorbed by them was
evaluated in vivo. For reaching the
aim of the study we have used peritoneal macrophages obtained from white
laboratory male mice 2 months of age, and weight - 22 ± 2 g. Total of 36
animals were randomized on 4 groups:1 Group – (Control) -
mice with NaCl solution (0,9 %) injection, (n=11), 2 Group – mice with dexamethasone
injection, (n=11), 3 Group - mice with dexamethasone and BCG injection, (n=11),
4 Group - mice with dexamethasone, BCG and formyl peptide injection, (n=11).
Animals were kept in vivarium of "Mechnikov institute of Microbiology and
Immunology of NAMS of Ukraine" on a standard diet with specified
conditions of animal management. Work with laboratory animals was performed
according to the rules. The peritoneum macrophages functional activity was
assessed by using Staphylococcus phagocytosis test, proliferative activity of
lymphocytes - by the level of their spontaneous and FGA-induced transformations
in vitro (RBTL), level of receptor’s expression on lymphocytes was examined in
reaction of lymphocytes rosette formation with sheep red blood cells. The number
of different types of cariocytes in the leukogram was counted morphologically
using the light microscope «PrimoStar» (Carl Zeiss, Germany), taking into account not less
than 200 cells in the preparation stained with Romanowsky-Gimza stain.
Statistical significance was determined by using unpaired t test
and one way ANOVA. p< 0,05 was taken as the level of
significance. Results and discussion The
introduction of the new formyl peptide to mice injected with BCG and
dexamethasone promoted increasing of the phagocytic activity of peritoneal
macrophages. What is demonstrated by significantly growing of phagocytic and
lytic indexes in this group compared to the group of mice that has not obtained
the formyl peptide after BCG and dexamethasone administration, (p<0,05).
Also, was observed the ex vivo
enhancing of FGA-stimulated (by 1,4 times) and spontaneous lymphocyte
transformation (by 1,8 times) after the formyl peptide’s administration
compared to the appropriate control group, (p<0,05). New formyl
peptide is able to promote the expression of receptors on lymphocytes. The
percent of receptors expression on lymphocytes has raised by 2,7 times after
the formyl peptide's administration in mice with BCG and dexamethasone
injections. The formyl peptide administration has led to the normalization of
blood cells count, when their depletion after dexamethasone and BCG injection
has taken place. Conclusions The new formyl peptide administration to mice
injected with BCG and dexamethasone promotes increasing of the phagocytic
activity of peritoneal macrophages, enhance the FGA-stimulated and spontaneous
lymphocyte transformation, enhances the level of receptors expression on
lymphocytes compared with to the group of mice that has not obtained the formyl
peptide after BCG and dexamethasone injection, (p<0,05). The formyl peptide administration normalizes the blood cells count
compared to the appropriate control group, where the total count of leucocytes
was decreased and ratio of neutrophils, lymphocytes and eosinophils were
characterized by a disproportion.
Key words: tuberculosis infection,
formyl-peptides,
immune system cells, phagocytosis, immunosuppression, experiment.
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62-66
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ANALYSIS OF THE
EFFECTIVENESS OF INDIVIDUALIZED TREATMENT REGIMENS FOR NONTUBERCULOUS
MYCOBACTERIOSES
Shevchenko O.S.,
Kalmykova I.M., Novohatska M.F.,
Shyrapova O.V., Pogorelova O.O.
Despite the fact that in Ukraine, as well as worldwide
the incidence of nontuberculous mycobacterioses is growing, in our country
there are still no standardized protocols for their diagnosis and treatment,
which makes it impossible to prescribe adequate chemotherapy and worsens the
prognosis of treatment. We have retrospectively studied medical histories of 26
patients who were diagnosed with "pulmonary non-tuberculous
mycobacteriosis" during 2014-2016. The diagnosis of "non-tuberculous
mycobacteriosis" was established based on the growth of non-tuberculous
mycobacteria (NTMB) in BACTEC system, and then verified by the absence of
Cord-factor formation, negative immunochromatographic test, negative GeneXpert
MTB / RIF. Based on the results of the studies, it was determined that 17
patients had slow-growing chromogenic NTMB and 9 patients had slow-growing
non-chromogenic NTMB. With the use of 2HRZE regimen, 83.3% of patients with
slow-growing chromogenic NTMB underwent laboratory recovery, and only 41.7% of
patients had clinical and X-ray recovery. However, patients who received the
individual regimen (2R(Rfb)Z(E)LfxKm) had a rapid positive dynamics, clinical,
radiological and laboratory recovery until the end of intensive phase. In
patients with slow-growing non-chromogenic NTMB, all three regimens (2HRZE,
2R(Rfb)Z(E)LfxKm, 2R(Rfb)Z(E)LfxClr) proved to be effective. We believe that it
is necessary to improve level of identification of NTMB for the timely
appointment of an adequate chemotherapy regimen.
Keywords. Nontuberculosis
mycobacterium, chemotherapy
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67-70
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SYNERGISTIC EFFECTS
OF ETHANOL MEDICINAL PLANT EXTRACTS WITH ERYTHROMYCIN AGAINST SKIN STRAINS OF
STAPHYLOCOCCI WITH INDUCIBLE PHENOTYPE OF MLS-RESISTANCE
Yurchyshyn O.I.,
Rusko H.V., Kutsyk R.V.
Introduction. One of the main ways
to control microorganisms’ resistance to antibiotics is to find substances that
are able to overcome it and potentiate antibiotics action, in particular to
neutralize the antibiotic-inactivating enzymes or block the active efflux of
antibiotic from microbial cells. Every year there is a growing interest in the
therapeutic potential of herbal active compounds as modifiers of antibiotic
resistance including MLS-resistance (macrolide-lincosamide-streptoramin B). It
should be emphasized that a number of biologically active substances of plant
origin can potentiate antimicrobial activity of erythromycin (ERY) against
MLS-resistant staphylococci. The present study was designed to investigate the
antibacterial and synergistic effects of eight Ukrainian ethanol medicinal
plant extracts with erythromycin against skin strains of staphylococci with
inducible phenotype of MLS-resistance. Material
& methods. S. aureus and S. epidermidis strains were tested for
susceptibility to antibiotics of MLS-group by disk diffusion test. Effective
antimicrobial concentrations of plant extracts and erythromycin were determined
by two-fold serial dilution in nutrient agar and broth. Combinatory effects
between organic extracts and ERY were assessed using the checkerboard assay
against tested strains to evaluate culture growth in the presence of two
antimicrobials with different concentrations. Results & discussion. The Alnus incana L. fruits extract was the most potent inhibitor against tested strains (MIC 40.625-162.5 µg/mL); while Geranium pratense L. rhizomes extract exhibited the least antimicrobial activity (MIC 650-2,600 µg/mL). The Alnus incana L. fruits extract and the Geranium pratense L. rhizomes extract
showed synergistic effect with erythromycin against 100% strains of
staphylococci (average FICI 0.028 – 0.057; p<0.001). In the presence of 1/4 MIC of ERY Alnus incana L. fruits extract antimicrobial concentration was decreased in
32-64 times and Geranium
pratense L. rhizomes extract
antimicrobial concentration was decreased in 64-256 times. Ethanol
extracts of Betula verrucosa L. buds (average
FICI 0.473±0.20), Arctostaphylos uva-ursi (L.) Spreng. leaves (average FICI 0.143±0.18) and Tamarix ramosissima Ledeb. leaves
(average FICI 0.189±0.29) showed synergic action with
erythromycin against 71.4-85.7% tested strains. Ethanol
extracts of Sanguisorba officinalis L. roots showed
non-interactive action with antibiotic against 42.8% isolates of staphylococci. Additive interaction with
erythromycin for this extract was observed against 28.6% and synergic action against 28.6% strains (average
FICI 0.812±0.52). Biota orientalis (L.) Endl. (Platycladus orientalis (L.) Franco) fruits extract and Cotinus coggygria Scop. (Rhus cotinus R.)
leaves extract exhibited non-interactive action with antibiotic against
all tested strains (average
FICI 2.0±0.0). Experimental data
indicate that combination of plant extracts with macrolides in therapeutic
regimens against MLS-resistant staphylococci is promising, particularly for the
treatment of pyoderma. The introduction of combined chemotherapy in clinical
practice can actually help to solve two problems of modern medicine - slow the
process of microorganisms (such as staphylococcus) resistance to antibiotics
acquiring and improve treatment of infections caused by resistant strains.
Detection of bacteria antibiotic resistance modifiers in various plants
stimulates to their intensive phytochemical study for isolation and
identification of the active components. It will help to investigate the
mechanisms of synergy on the molecular level. Conclusion. BAC of medicinal and aromatic plants potentiate
antimicrobial activity of macrolides against skin isolates of staphylococci
with inducible MLS-resistance. Alnus
incana L. fruits ethanolic extract demonstrates the best direct
antimicrobial activity and in combination with ERY synergistically inhibits the
growth of S. aureus and S. epidermidis MLS-resistant strains. Ethanolic extracts of Geranium pratense L. rhizomes, Arctostaphylos uva-ursi (L.) Spreng.
leaves, Tamarix ramosissima Ledeb.
leaves and Betula verrucosa L. buds
also exhibit synergistic effect with ERY against skin isolates of staphylococci
with inducible MLS-resistance.
Keywords: plant extracts, erythromycin, staphylococci,
MLS-resistance, synergistic effects.
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71-79
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