The  review  article  considers the  data  from  literature that  concern polymorbidity aspects,  its interrelations with ageing of immune system and lo-grade immune ageing, mechanisms of genesis, approaches to its prevention and treatment. Evolution of “comorbidity” and “polymorbidity” terms is traced, an updated definition of polymorbidity is proposed. The  world-wide incidence of polymorbidity is increased and  now it reaches  23-25%  in general  population, and up to 98%, in elderly people  (> 65 years old).  The risk factors  of polymorbidity are considered, like as its social burden due to high costs for healthcare, high mortality rates, excessive treatment provided by multidisciplinary specialists.  We present  evidence  for common molecular and cellular  mechanisms involved  in ageing and polymorbidity, being unified  by the term  “inflammaging” which represents a low-grade chronic systemic  inflammation associated with  ageing.  The  data  are  presented that concern the “inflammaging” development with involvement of ageing cells from innate and adaptive immunity systems,  different   pro and  anti-inflammatory mediators, lifelong  antigenic load.  The  data  are  analyzed concerning functional and  structural changes  in the  inborn and  adaptive  immune system  in ageing,  role of these changes  in “inflammaging” persistence and development of polymorbid conditions. There  are complex interactions shown between  the bodily senescence and immune ageing, with similar underlying mechanisms in some cases, however, being quite different  in other  instances. With age, upon  existing risk factors,  the changed adaptive  immunity in most people  is not able to full-scale  coping  with chronic antigenic load,  thus increasing the risk of diseases. Moreover, in many elderly people these changes are compensated by steady activation of the innate immunity cells. It is noted  that the aging events and development of disease (polymorbidity) cannot be considered distinct entities, since they can interact, being, however, basically different in their nature. In future, one should concentrate our efforts on elucidation of molecular and cellular mechanisms of these interactions, solution of the  tasks  oriented for development of such  interventions that  could  be able  to  reduce  harmful consequences of ageing and to use useful effects for health  maintenance and reaching maximal longevity.

Macrophages (Mφ) play a key role in regulation of fibrogenesis, including proliferation of fibroblasts and  myofibroblasts, differentiation of progenitor cells into  myofibroblasts, as well as synthesis  and  secretion of the  extracellular matrix, mainly  collagen.  The  direction of the  Mφ effects (stimulation or suppression)  is determined by a number of factors,  including the stage of the fibrotic process and the Mφ functional phenotype dependent on the signals of microenvironment. One  of the feasible ways of the fibrogenesis  regulating is the secretion of pro-  or antifibrotic factors such as matrix metalloproteinases, inhibitors of metalloproteinases and some cytokines. However, existing data on ability to secrete  these factors by various subpopulations of human Mφ are rare and controversial. The aim of this study was to characterize the ability of human M1,  M2a,  and M2c Mφ differentiating in the presence of GM-CSF to produce matrix metalloproteinases (MMP-9) and their tissue inhibitors (TIMP-1), as well as some cytokines and growth factors. As compared to M2 macrophages, the M1 macrophages polarized by lipopolysaccharide produced significantly  more TNFα, IL-6 and IL-2 that have pro-inflammatory activity and are able to initiate a fibrotic process.  In turn,  M2a Mφ stimulated by IL-4  were characterized by a high level of VEGF production and, at the same time, low levels of TNFα and IL-6, which may determine the important role of these cells at the proliferative stage of fibrosis and stimulation of extracellular matrix deposition. Finally, M2c Mφ polarized by dexamethasone, exhibited  the М2а-like cytokine profile, i.e., VEGF was actively produced against the background of low TNFα and IL-6 synthesis.  Moreover, all three Mφ subpopulations did actively secrete MMP-9 and TIMP-1, without significant  difference in production of these factors. However, M2c Mφ differed by a significantly  higher MMP-9/TIMP-1 ratio index compared to M1 and M2a Mφ, and it is crucial  at the rearrangement stage of the fibrotic  process.  Thus,  the production of MMP-9 and TIMP-1, together with other  pleiotropic cytokines and growth factors by various Mφ subtypes may reflect their role in regulation of fibrotic process at various stages.

Extracellular vesicles that are shed from the plasma membrane contain a wide range of molecules, among  which  are proteins, lipids, nucleic  acids,  and sugars. The cytotoxic proteins of natural killer cells play a key role in the implementation of their cytolytic  functions. One of the important steps in understanding the distant  communication of cells is the determination of the proteome of microvesicles. This study was aimed at the protein profiling of the microvesicles produced by the NK-92 natural killer cell line. 986 proteins with a variety of functions were identified in the lysate of microvesicles using the MALDI-TOF mass spectrometric analysis.  With automated methods of functional analysis  applied, it has been  shown  that  the  largest  protein groups  are  hypothetical proteins, proteins with  unknown functions, and  domains. The  most  representative groups  are  also  comprised by  transcription  regulators; intracellular  signaling  proteins; RNA  translation, transcription, processing, and utilization regulators; receptors; protein processing  and proteolysis regulators; amino acid metabolism enzymes, as well as transport proteins and transport regulators. Minor functional groups are represented by vitamins and mineral metabolism enzymes, membrane and microdomain-forming proteins, hormones, hemostatic regulators, regulators of sensory  systems,  specific  mitochondrial and  Golgi  apparatus proteins, and extracellular signaling proteins. An intermediate position is occupied by various functional groups, including cytoskeleton and motor proteins; proteins of centrioles; ion channels and their regulators; proteins of the ubiquitin-proteasome pathway  of protein degradation; lipid,  steroid, and fatty acid metabolism enzymes; nucleic  acid  base and  carbohydrate metabolism enzymes, as well as energy  metabolism enzymes  and  other proteins involved  in intermediate metabolism; proteins of the immune response  and  inflammation; antigens and histocompatibility proteins; cytokines and growth factors; regulators of apoptosis, autophagy, endocytosis, and  exocytosis;  regulators of the  cell cycle and  division;  regulators of proliferation, cell differentiation, and morphogenesis; regulators of cell adhesion and  matrix  metabolism; nuclear transport proteins; transposition proteins; DNA  replication and  repair  proteins, as well as inactive  proteins. The  data  obtained expand  the existing knowledge of the distant  communication of cells and indicate new mechanisms of interaction between natural killer and target cells.

Experimental traumatic brain  injury  (TBI)   causes  a  stable  stress  response   and  changes   the expression  of various cytokine genes and neurotrophic factors.  The goal of this study was to reveal changes  in the  levels of the  corticosterone and  testosterone hormones and  the  BDNF cytokine in blood  serum,  as well as the expression  of the BDNF gene in hypothalamus in order  to determine the opportunity of correcting the TBI damage  with rIL-2. We used a rat model  of “dropping load”:  mild TBI was caused  by falling of the 115 g load from the height  of 80 cm,  or 120 cm to produce a moderate-degree trauma. After TBI (immediately, or 72 hours  later), the  rats were injected daily with recombinant human interleukin-2 (Roncoleukin) at a dose of 30 μg/kg, a total  of 3 injections. Control animals  (also with TBI)  received  0.15 M NaCl  injections. Blood serum  concentrations of corticosterone, testosterone, and  BDNF were measured with ELISA  tests.  BDNF gene expression in hypothalamus was measured using RT-PCR. Results: the experiments showed a relationship between  hormone concentrations and severity of head injury. In mild TBI,  blood corticosterone levels reached a peak  2 hours  after the  injury, while in moderate TBI,  the  peak  concentration of corticosterone was lower, being delayed  in time  (after  24 hours). Corticosterone and  testosterone concentrations changed reciprocally in the both groups of injured  animals. With injection of rIL-2 in both groups,  corticosterone and testosterone levels were significantly  increased. On  day 7 after  TBI,  the  BDNF level in blood  serum  was decreased, but it was raised  in experimental group  that  received  rIL-2. On day 7, the increase  of BDNF gene expression  in hypothalamus was more  pronounced, when  rIL-2 was administered at 72 hours  after  the  head  injury.  The revealed  positive  association of BDNF levels and  glucocorticoid hormones after  mild  TBI,  like as possible coordination of these  parameters with rIL-2 injection after experimental moderate TBI  provides  a reason  to assume  that  the favorable  impact of rIL-2 on the CNS  recovery  after TBI is, in part,  mediated by the mutual modulating interaction of BDNF and glucocorticoid hormones.

The aim of our research  was to reveal quantitative ratios existing between  the pathways of cellular death in normal state, as well as in immunocomplex pathology. The proportion of different pathways of cell death (autophagy, apoptosis, necrosis) in autoimmune (systemic  connective tissue diseases (SDCT) – rheumatoid arthritis (RA), systemic lupus erythematosus (SLE)  and systemic scleroderma (SSD) is a subject of age-related changes. On the one hand, aging process can be considered a genetically determined overall decrease in adaptive potential of the body, and a systemic  age-related chronic inflammatory response, with a pronounced cytokine proinflammatory shift. On the other hand, a polygenic decrease in energy and information capacity  of the cells, represent the basis of multisystem and multiorgan functional and metabolic disorders  in SDCT.

Blood plasma  samples were analyzed  in the patients of two age groups. The first group consisted of 10 SLE cases (4 men and 6 women, average age 43.8 years), 13 patients with RA (5 men and 8 women, average age 45.6 years),  7 SSD (women, average age 35.8 years),  and 10 healthy  donors  (6 men and 4 women, average age 40.7 years). The second age group consisted of 9 SLE cases (2 men and 7 women, average age 69.8 years), 10 patients with RA (5 men and 5 women, average age 65.6 years), 5 patients with SSD (women, average age 65.7 years) and 12 healthy  donors  (normal biological  aging – 7 men and 5 women, average age 64.7 years). The data presented in this paper  were obtained with informed consent of the  patients. When  carrying  out  biomedical research, we followed  internationally recognized ethical  standards of the Helsinki Declaration (International Medical Association, 1996, revision  2013). The proportion of various cell death  types (autophagy, apoptosis, necrosis) in  autoimmune disorders  (systemic  diseases  of connective tissue,  SDCT), i.e.,  rheumatoid arthritis (RA), systemic  lupus erythematosus (SLE), and systemic  scleroderma (SSD) proved to be subject to age-dependent changes. Close  interaction were revealed  between  the  ways of cellular  death  in SDCT (most  pronounced in SLE), correlating with age changes and clinical  manifestations of autoimmune process. In SDCT, the affected tissues exhibit all types of cellular  death, however, degree of their expression  depends on the disease nosology. Upon systemic diffuse pathology of connective tissue, autophagy (especially in case of SLE and RA) is directly involved in development of immune response  and inflammatory process.

In normal biological  aging, like as in SDCT, one may observe a sharply increased activity of the metabolic trigger – AMP-activated protein kinase (AMPK), a sensor of intracellular energy, along with shifted acid-base equilibrium. The quantity of active oxygen radicals  increases, oxidoreductive potential of the cells is changed, with  activation of cellular  destruction components. Activity  of cytokine system  in the  organism  is changed causing  apoptosis regulation; expression  of chaperons is decreased, and  the immune-oxygenase homeostasis is also displaced. Inhibition of genetically determined process of death  of cells (apoptosis) comprises the basis for development of autoimmune diseases. Transition of late apoptosis into secondary necrosis  is accompanied by decrease of antioxidant protection and  development of autoimmune pathology. The  chaperon-mediated induction of immune response as the signaling mechanism of autophagy, being evolutionarily fixed in mammals only, may be the common central  link and “the  molecular switch”  causing both development of autoimmune diseases of connective tissue, and aging processes.

Pathogenesis of ischemic stroke  is actively  involved  in the  system  of innate immunity. Under conditions of cerebral  ischemia, a number of biologically  active  substances are  released  that  interact with innate immunity receptors, in particular TLR2  and  TLR4, which  exacerbate inflammation in brain  tissue. Identification of predictor markers  at the level of the innate immunity system may foresee the clinical course of ischemic stroke and ensure timely treatment. Our objective was to study expression of TLR2 and TLR4 receptors in peripheral blood leukocytes  in patients with ischemic stroke in the dynamics of the disease. 27 people  were included in the study. The main  group consisted of patients with ischemic stroke of varying severity (n = 19). Patients of the main  group were divided into two subgroups:  with an NIHSS index value of < 10 (n = 10) and > 10 (n = 9). The control group included healthy  donors  with no history  of acute  and chronic inflammatory diseases (n = 8). Peripheral blood  leukocytes  were used as the  test material. To determine expression  of the TLR2  and TLR4  genes, RT-PCR in real time was used. Surface  expression  of TLRs was determined by flow cytometry. A study of the TLR2 and TLR4 gene expression showed that on the 1^st, 3^rd  and 7^th  day post-stroke, the TLR4 gene expression  in patients was significantly  increased, when compared to the control group (p < 0.01), whereas TLR2 gene expression on the 3rd  day of the disease was not statistically different from the control group. A study of surface expression  of receptors showed that the average TLR2 fluorescence intensity on the patients’ peripheral blood monocytes was significantly  increased on the 1st  and 3rd  day of disease when compared to the control group.  The  surface  expression  of TLR4  on monocytes has a statistically significant  increase  only on day 7. Assessment  of surface expression  of TLRs in subgroups  with different  severity values by NIHSS showed that  patients with a NIHSS index > 10 had a significantly  higher  level of surface of TLR2  expression  over the observation period, while the largest difference in TLR4  expression  in the subgroups  was observed  on the 1st day of the disease (p < 0.05). Patients with ischemic stroke showed an increase  in TLR2 and TLR4 expression at the gene and protein level, compared to healthy  donors. These indices can be considered possible predictors for clinical  prognosis  of ischemic stroke.

Early prediction for ischemic stroke (IS) outcome is a major challenge since it may help to optimize treatment program and  to make  it more  personalized. Since  T cells with  regulatory activity  are involved  in different  pathophysiological processes  in brain  stroke,  including inflammation, immune suppression, brain damage  and  repair, the  study  of T cells as potential biomarkers has essential  importance. The  present  work aimed to study the circulating T cell subsets with phenotype of type 1 T helper cells (Th1) and regulatory T cells (Treg), and their  ratio during  the acute  phase of IS, depending on stroke severity, inflammatory response  and 3-month outcome (according to modified Rankin scale, mRs).  Patients and methods. The study included 61 patients with a newly diagnosed IS (severity according to NIHSS ≥ 5), in the first 24-48 h after stroke onset, and 20 age/sex-related healthy  donors. Laboratory examination included assessment of leukocytosis, neutrophillymphocyte ratio  (NLR) and CRP  concentration. Mononuclear cells were isolated  from peripheral blood  to study T cell subsets. Th1 and Tregs were measured by FACS  analysis as CD4⁺IFNγ⁺  and CD4⁺CD25^hiT cells, respectively. During the first 24-48 h after stroke, the patients had elevated values of leukocyte counts, NLR and CRP. Higher  levels of these parameters in severe stroke compared with mild stroke, as well as direct correlation of NIHSS with  NLR  and  CRP  evidenced that  the  stroke  severity  was associated with  more  pronounced inflammatory response. Patients were also characterized by a significant  decrease in CD4⁺IFNγ⁺Th1  cells, an  increase  in CD4⁺CD25^hiTreg, and  a marked  decrease in Th1/Treg ratio.  Furthermore, in patients with NIHSS ≥ 8 (moderate and severe stroke), the percentage of CD4⁺IFNγ⁺T cells was in direct  correlation, and the number of CD4⁺CD25^hiT cells was inversely related to CRP  and NLR  values. The changes of T cell subsets were more  pronounced in patients with  a favorable  3-month outcome (mRs  > 3). As a result,  the  patients with poor outcome (mRs  ≤ 3) had higher  CD4⁺IFNγ⁺T cell proportion, lower CD4⁺CD25^hiT cell percentage and  4-fold  higher  CD4⁺IFNγ⁺/CD4⁺CD25^hi  ratio  compared with opposing  group.  ROC  analysis  revealed  a “good” quality of prognosis  based on evaluation of the CD4⁺IFNγ⁺/CD4+CD25^hi  ratio as a monopredictor of adverse outcome (AUC  = 0.75) and “very good”  quality  of prognosis  when the indicated ratio was combined with  NIHSS scale  (AUC  = 0.82).  The  data  obtained suggest  that  a decrease of Th1/Тreg ratio,  due  to  a decrease in CD4+IFNγ+  and increased CD4⁺CD25^hiT cell counts  during the acute  phase of ischemic stroke is a compensatory reaction directed at inhibition of inflammatory response, and has a prognostic significance as early predictor of the outcome at 3 months.

Chronic pain in humans remains a challenge for diagnosis.  It manifests itself as multicomponent symptoms and leads to dysregulation of many  biochemical systems.  The approaches based on measuring the neurohumoral factors  regulating transmission of a pain  signal,  are promising for the  pain  evaluation. These include  immunological parameters, such  as natural antibodies (e-At), which  can  specifically  interact with endogenous bioregulators of pain  impulse  (EB), especially, with  serotonin, dopamine, and  modulate the process of pain development. Antibody metabolism is characterized by longer circulation in the bloodstream as compared to EB. Therefore, the content of e-At to EB reflects long-term changes in the body upon development of chronic pain.  Detection of relationships between  their  level and  the  course  of treatment will allow us to establish the prognostic role of immunological parameters in objective assessment of pain status of patients.

The study included 136 patients (70 women  and 66 men)  with chronic pain syndrome. The patients were subjected to assays of e-At to dopamine, serotonin, and a survey using a visual analogue scale, in order to assess the intensity of pain. The indexes were measured in the course of treatment (1st, 10th  , and 21st  days).

As a result,  a significant  decrease in pain intensity was found  in 63% of women  and in 71% of males. E-At levels in patients admitted for treatment were initially mostly elevated  and high. The dynamics of e-At change was multidirectional. On the day 21, an increase  in the occurrence of normal levels of e-At  to serotonin was detected in 52% of women  and in 59% of men.  The content of e-At to dopamine in this period  was recorded at a normal level in 56% of women, however, being increased in men (50% of cases), with high levels in 17% of males.

Thus,  examination of patients with CHD showed  that,  against  the  background of ongoing  therapy, pain intensity decreases, and antibodies to pain mediators may continue to circulate at elevated  concentrations. It is likely that the body maintains pathologically elevated levels of e-At to EB, reflecting the content of EB itself, contributes to prolongation of CHD. Monitoring individual profile of the immunological parameters of e-Ab to EB in patients may have prognostic value for choosing an effective,  personalized treatment program.

Joint  damage  initiates aseptic  self-sustaining inflammation, which  contributes the  progression of post-traumatic destruction of tissues  not  only  in the  pathological focus,  but  also outside  it,  significantly expanding the zone of degenerative changes due to secondary alterations. One of the leading roles in pathogenesis of the inflammation belongs  to secreted  mediators-cytokines – that  impart to the cells the proinflammatory potential and  promote the  long-term inflammation. These  effects  lead  to  disorganization of extracellular matrix and progressive  disintegration of cartilage.  In this regard,  the development and implementation of new pathogenetic treatment methods of post-traumatic synovitis permits  to limit the area of secondary alterations and activate  reparative mechanisms in the lesion  from the early terms,  thus potentially improving the results of  rehabilitation treatment and  increasing efficiency  of  conventional therapy   in  post-traumatic synovitis.

Numerous experimental and  clinical  studies  have proven  the  effectiveness and  safety of ozone  therapy, e.g., in degenerative joint  diseases.  Despite extensive  data  highlighting effectiveness of ozone  therapy  in articular pathology, the  study of cytokine profile  when  using this treatment of posttraumatic synovitis  was performed only in few works, thus emphasizing the prospects for further research in this direction. The study was aimed for investigation of cytokine status in the patients with posttraumatic synovitis subjected to intravenous and intraarticular ozone  therapy  in combination with intra-articular administration of xefocam.

The  work is based  on  the  results  of examination and  treatment of 69 patients with  traumatic injuries  of the  knee  joint,  complicated by development of  post-traumatic synovitis.  Two  study  groups  were  formed, comparable in volume  and  type  of joint  injury.  The  patients from  group  I (35 cases)  received  conventional combined treatment. Among  the  mandatory measures, evacuation of a synovial-hemorrhagic punctate was performed from the cavity of damaged joint. Conservative therapy included NSAIDs, medications that improve microcirculation, at standard dosages, as well as physical therapy. In group II (34 patients), traditional therapy was supplemented with a 10-day  course of intravenous injectable ozone  therapy  with 200 ml of NaCl  solution at a concentration of 2.0 mg/l daily and intra-articular ozone injection at a concentration of 5 mg/l in a volume of 20 ml 5 times  in a day. During arthroscopy, lavage of the joint  cavity was performed with ozonated saline solution at a concentration of 2.0 mg/l.  The ozone  therapy  was combined with three  intra-articular injections of xefocam  at a dose of 8 mg, once  every 4 days. A patent for the  invention was obtained for this treatment technology (No.  2456988 of 27.07.12).  The cytokine profile was evaluated by the content of Pro-inflammatory (TNFα, IL-1β, IL-6, IL-17), regulatory (IL-2), Il-1β receptor antagonist, and anti-inflammatory (IL-4, IL-10) cytokines by solid-phase enzyme  immunoassay with an indicator label in the  form  of peroxidase. Statistical analysis of the results was carried  out using the Student criterion. Combined therapy  of intravenous and intraarticular ozone therapy  in combination with intra-articular injections of xefocam  contributed to the inhibition of the  inflammatory response, which  is reflected in  the  dynamics of depression of the  studied  cytokines: simultaneous reduction of proinflammatory cytokines with the limitation of the growth of anti-inflammatory mediators. The final measurements showed a decrease in the content of proinflammatory cytokines: TNFα by 24.6% (p₂   < 0.001);  IL-17, by 17.3% (p₂   < 0.01);  IL-6, by 20.1% (p₂   < 0.001);  IL-1β, by 19.1% (p₂   < 0.001), with a decrease in regulatory IL-2  by 25.7% (p₂   < 0.001) and anti-inflammatory cytokines IL–10, by 21.3% (p₂  < 0.001); Il – 4, by 25.7% (p₂  < 0.001); IL-1ra, by 24.4% (p₂  < 0.001), when compared to the data obtained with conventional treatment. The  results  obtained allow us to evaluate  this method as highly effective  in the treatment of post-traumatic synovitis,  thus contributing to suppression of inflammatory response  and reduces the secondary alteration of joint tissue structures, preventing the progression of post-traumatic osteoarthritis.

According to new views on communication ways and  principles in the main  regulatory systems of the  body, i.e.,  immune and  neuroendocrine, there  is a risk for disintegration of pathways  and  structures in these  systems which  may underlie disorders  such as autism-spectrum disorders  (ASD)  and schizophreniaspectrum disorders (SSD). Both disorders are classified as neurodevelopmental disorders, with unclear etiology and partially  overlapping pathophysiological developmental mechanisms. Diagnosis of ASD and SSD is based on patterns of clinical  symptoms/syndromes that  demonstrate high heterogeneity and  similarity.  Therefore, it is very important to find the  ways of discerning children with ASD from  those  with SSD.  Our  aim was to identify peripheral activity indexes for immune and neuroendocrine systems, and their integration for usage as information hubs of congruency and phenotypic plasticity of these systems in children with ASD, as compared to SSD  patients. The  levels of 14 indexes  of the immune and  neuroendocrine systems in blood  plasma  were determined in 82 children with ASD, 9 children with SSD and 45 children with typical neurodevelopment (TD). To assess peripheral activity of the immune and neuroendocrine systems and their relationships, we applied  a multivariate exploratory analysis using a method of nonlinear principal components. The following results were obtained: (1) absence  of differences in proinflammatory cytokines between  ASD and TD children; (2) patients with SSD  have significantly  higher  values of IL-6  and IFNγ, and lower values of IL-1β, TNFα and IL-10  in blood plasma compared to children with ASD and TRD; (3) the level of neurohormones in children with ASD is in accordance with physiological reference values. The children with SSD have lower levels of epynephrine and dopamine compared to ASD and TD,  respectively; (4) integration degree of regulatory systems assessed by principal component analysis has shown  the following:  (4.1)  TD  children have strong  correlations within each of the systems and between  them, thus showing their communicative abilities and plasticity, characteristic of normal values; (4.2) In SSD  children, minimal numbers of strong  relations were demonstrated within  the cytokine system;  (4.3)  The children with ASD exhibited  two clusters:  one of them  had a complete similarity with TDC, in terms of tension and assortment of immune and neuroendocrine indices; the other one presented low coupling between  the parameters of regulatory systems, similar to the children with SSD; (4.4) Analysis of peripheral indices of cytokine and neuroendocrine systems for clusters 1 and 2 in children with ASD compared to children with SSD and TD demonstrated that,  in children with ASD of cluster  1, the indices  did not differ from TDC, except  of epinephrine, ACTH, kynurenine, and tryptophan. In the children with ASD of cluster 2, the values of the indices are equal to children with SSD,  except of dopamine and tryptophan. Thus,  we have shown phenomenon of transdiagnostic clustering, i.e., allocation of two clusters among  ASD children. One of them is similar to levels of indices and connections between the immune and neuroendocrine systems with TD, and another cluster is similar to SSD children. Therefore, they could be potentially useful as diagnostic criteria when discriminating the two disorders.

Inflammatory bowel diseases (IBD), such  as Crohn’s disease (CD) and  ulcerative colitis (UC), are characterized by chronically recurring inflammation of intestinal wall and are associated with a significant decrease in the  quality  of life. A spectrum of genetic  variants  associated with  Crohn’s disease  is described. Intestinal dysbiosis (DB)  may be the triggering factor of the disease. Glycoprotein 2 (GP2), the main protein of pancreatic zymogen  granules, is secreted  into the intestines with digestive enzymes.  Anti-GP2 antibodies were found in the serum of patients with CD.  The aim of the present  study was to investigate  the levels of anti-GP2 antibodies in serum  and feces of children with IBD  compared with the DB group.  Serums  and coprofiltrates from 110 children (64 boys and 46 girls) at the age of 12.3 (2.6-17.9) years were studied; 36 patients with CD, 30 patients with UC.  A comparison group consisted of 44 patients with DB. IgG and IgA antibodies against GP2 were tested with ELISA. Nonparametric statistics methods are applied, the results are presented as percentages and medians (Me (Q_0.25-Q_0.75)). The serum levels of anti-GP2 IgA antibodies were 9.97 (3.35-13.45) U/ml for the CD patients, 6.08 (2.71-14.26) U/ml for UC and 2. 94 (2.29-6.41) U/ml for DB. The levels of anti-GP2 IgG antibodies in serum were 6.16 (3.26-18.4) U/ml for CD, 5.26 (2.97-7.52) U/ml for UC, and for DB 5.23 (2.53-8.85) U/ml. The cut-off  threshold concentration for anti-GP2 IgG antibodies was 13.8 U/ml, with sensitivity of 63.2%, specificity 100%, and for IgA 5.63 U/ml, with sensitivity of 60.5% and specificity of 78.8%, thus being lower than the calculated cut-off  for adults (20 U/ml). The levels of anti-GP2 IgG in coprofiltrates in children of comparison group  were 1.99 (1.26-3.04) U/ml; in the  patients with CD, 23.5 (16.15-29.3) U/ml, and  in children with UC, 20.45 (13.63-25.5) units/ml (p < 0.001). The cut-off  value amounted 8.0 U/ml, with 100% sensitivity  and  100% specificity.  Concentrations of anti-GP2 IgA in coprofiltrates of patients with IBD  did not significantly  differ from DB patients. Moreover, the concentration of sIgA in the coprofiltrates of patients with IBD  was significantly  higher than  their level in DB group. The anti-GP2 IgA/sIgA  ratio was significantly lower in patients with CD (0.326 (0.23-0.512)), and UC (0.327 (0.205-0.435)), than in patients with DB (2.332 (1.575-3.523)) (p < 0.001);  the cut-off  level was 0.784, with a sensitivity of 97.7% and specificity  of 98.6%. It is discussed, whether fecal anti-GP2 IgA antibodies should  be considered as protective, supporting intestinal homeostasis, whereas anti-GP2 IgG antibodies are pathogenetically significant  for development of IBD.  Thus, using a non-invasive method for determining anti-GP2 antibodies in stool, when exceeding the cut-off for IgG, and reduction of IgA/sIgA ratio below the cut-off, one may differentiate IBD from DB with a similar symptoms at the onset of disease, with 100% sensitivity and 100% specificity.

The  aim of the study was to evaluate  the effect of sodium  deoxyribonucleate on anti-infectious resistance and hematopoiesis in patients with polytraumas. A single-center study of sodium  deoxyribonucleate effectiveness approved by the local Ethics Committee (protocol No. 4 05/18/2016), was conducted in 54 patients with polytrauma. The  main  group  included 27 people, at the mean  age of 39 (29-51)  years old; ISS severity score,  26 (22-34). The  comparison group  comprised 27 people, mean  age,  40 years old (26-53), mean  ISS severity score was 25 points (20 to 29). The patients with randomly attributed even numbers were injected with 5 ml preparation from vials of even-numbered series, the patients with odd numbers were treated with preparation from the odd-numbered series. They were injected intramuscularly daily from day 1 to day 10 after the injury. Before treatment, as well as on days 8, 15 after injury, peripheral blood was examined for leukocyte, erythrocyte counts, hemoglobin, total  protein, blood  IL-6, CRP; proportion of CD117⁺  and  CD34⁺  mononuclear cells, CD14⁺ monocytes, CD14⁺ granulocytes, HLA-DR⁺  mononuclear cells, defensin + granulocytes.

On  the  day  +8,   patients from  the  main  group,  against  the  comparison group  showed  an  increase   in lymphocytes, monocytes, CD117⁺ and CD34⁺ cell counts. Serum IL-6 and CRP were decreased in both groups of the patients to a similar  degree. Terms  of hospitalization in the main  group were 32.8 days, against  39.6 in comparison group. The number of complications per 1 case was, respectively, 21 versus 39, thus being 1.8 times less than  in comparison group.  When developing complications, anemia (Hb  < 90 g/l),  or hypoproteinaemia (< 60 g/l) in the main group was, respectively, 2.5and 3.5-fold  less than  in the comparison group.

Treatment with sodium  deoxyribonucleate in polytrauma may promote migration of blood precursors to the bloodstream, increase anti-infectious properties of leukocytes, reduce duration of anemia and hypoproteinemia, number of complications and decrease the terms of hospitalization.

Exudative otitis  media  in  childhood is most  often  associated with  chronic inflammation in the  nasopharyngeal area,  with  immediate participation of phagocytic cells. Our  paper  presents  the  data  on evaluation of clinical  and immunological efficacy of intranasal Imunofan use included into  complex therapy of exudative  otitis  media.  Dynamic observation (before  treatment, 1 and  3 months after treatment) of these parameters included regular  evaluation of the  neutrophil and  monocyte amounts in peripheral blood  and  in smear imprints from nasal mucosa, determination of myeloperoxidase activity in circulating neutrophils, and the content of interleukin IL-8 and IL-18  in the nasal washouts. The clinical status was assessed using a scoring system, which subjectively reflected the state of the nasopharynx and auditory function. Fourty-three children aged from 3 to 7 years with exudative  otitis media associated with chronic adenoiditis were examined. Patients of the first group (22 children) were treated using only conventional approaches (basic therapy). The patients from  the  second  group  (21 children) received  Imunofan in addition to the  basic therapy. The  control group consisted of 16 relatively  healthy  children. Before  treatment of the  children with exudative  otitis  media, an increase  in the relative content of monocytes in their blood,  a decreased activity of myeloperoxidase and lower concentration of IL-8  and  IL-18  in the  nasal  wash was observed  in comparison with  healthy  controls. No differences in severity  of clinical  symptoms were revealed  between  the  groups  of patients. Baseline  therapy was not  accompanied by positive  dynamics in the  clinical  pattern of the  disease.  Relative  monocytosis and reduced activity of neutrophilic myeloperoxidase persisted  in peripheral blood;  the concentration of IL-8  and IL-18  in the  nasal washings  remained low. Following intranasal use of Imunofan, the  number of circulating monocytes was restored by the  third  month from  the  start  of treatment, there  was an  increased activity  of myeloperoxidase registered  in blood neutrophils, as well as higher IL-8  and IL-18  concentrations in the nasal washings. Normalization of the phagocytos-related parameters, according to this scoring,  was associated with clinical remission of the disease. The revealed relationships between clinical data and the results obtained in the course  of laboratory research  suggest a positive effect of Imunofan as an agent that may enhance effectiveness of conventional basic therapy  of otitis media in children.

We aimed  for assessing effects of immunocytotherapy upon  the subpopulations of CD4⁺CD25^highFoxP3⁺ cellswithnaturalregulatoryactivityandactivatedTh17cellswiththeCD4⁺CD25^highRORγt⁺ phenotype, as well as in vitro production of cytokines in mitogen-stimulated cells from  peripheral blood  in the patients with idiopathic habitual miscarriage (IHM). The study group consisted of 33 patients with IHM who became pregnant after a pre-gestational alloimmunization. In 27 patients, the pregnancy was prolonged to the  full term  and  ended  with the  birth  of viable babies,  in six cases it was terminated before  12 weeks of gestation. Before  administration of immunocytotherapy (ICT), 19 patients were examined, of them  16 after alloimmunization outside of pregnancy, 17 at 5-6 and 8-9 weeks of pregnancy. Eleven patients were immunized at 12 weeks of pregnancy. In the control group,  12 fertile women  outside  pregnancy and 10 women  at 12 weeks of physiological pregnancy were examined. The proportion of FoxP3⁺ and RORγt⁺ cells with the CD4⁺CD25^high phenotype was evaluated among  T-lymphocytes from peripheral blood,  as well as content of proinflammatory cytokines (IFNγ,  TNFα, IL-1β, IL-2, IL-5, IL  -6,  IL-8, IL-12p70) and  anti-inflammatory factors  (IL-4, IL-10), as well as IL-17  amounts.

We have found  that,  following pre-gestational alloimmunization, the women  who lost this pregnancy, had a  low  level  of  FoxP3+Тregs that  suppress  pro-inflammatory Th17-dependent  reactions, however, without changing levels of activated Th17  cells (CD4⁺CD25^highRORγt⁺   lymphocytes). These  facts,  along  with  high in vitro production of IL-17  by peripheral blood cells at the terms of 5-6 weeks of gestation, suggest that,  after pre-gestational alloimmunization in women  with miscarriage, a predilection is formed  to pro-inflammatory cytokine production. However, at the 5-6 week-period, it is realized  not in the Th1 direction of, but towards Th17 response, and a low level of CD4⁺CD25^highRORγt⁺ cells may reflect an increased migration of Th17 cells from peripheral blood to the uterine endometrium.

Thus,  we have shown  the  effect of immunocytotherapy upon  subpopulational composition of peripheral blood  lymphocytes and  the  cytokine profile,  as well as upon  the  course  of first trimester and  outcomes of pregnancy in women  with idiopathic habitual miscarriage.

The cytokine system is a large group of humoral factors  produced by immune cells and involved in the  pathogenesis of most  human diseases.  To assess the  significance of changes  in cytokines/chemokines under  pathological conditions, appropriate reference values are required for healthy  people.  As known  from existing literature, most studies of various cytokine/chemokine concentrations in blood plasma were performed in healthy  subjects from Western Europe and North America.  Certain inter-population differences are known, with  respect  to production of distinct cytokines in different  racial  and  national groups.  Only  single studies concern normal levels of distinct cytokines in blood  plasma  of healthy  African  residents. The purpose  of this study was to determine the blood plasma cytokine profile in healthy  residents of the Republic of Guinea (RG), and to establish normal cytokine values.

We have  examined 24  healthy  RG  residents and  23  residents of St.  Petersburg. Concentrations  of 40 cytokines/chemokines were determined in blood plasma.  The study was performed using multiplex analysis by xMAP technology.

The  following  cytokine/chemokine levels  were  significantly   increased in  the  blood  plasma  of the  RG residents: IFNγ, IL-2, IL-4, IL-6, IL-10, TNFα, CCL1/I-309, CCL3/MIP-1α, CCL7/MCP-3, CCL17/ TARC, CCL19/MIP-3β,  CCL20/MIP-3α,  CCL21/6Ckine, CXCL2/Gro-β,  CXCL5/ENA-78, CXCL6/ GCP-2, CXCL9/MiG, CX3CL1/Fractalkine (р < 0.001).  For  the  CCL8/MCP-2, CCL22/MDC, CXCL1/ Gro-α and CXCL12/SDF-1α+β chemokines a trend for increased concentration was revealed, in comparison with residents of St. Petersburg (р < 0.05). Moreover, the levels of CCL23/MPIF-1 and MIF were significantly lower (р < 0.0001) in the RG residents. There was a tendency for decreased levels (р < 0.05) for CCL2/MCP-1 and CCL24/Eotaxin-2 chemokines in blood plasma taken from RG residents. There were no differences in levels of cytokines/chemokines for the studied  groups: GM-CSF, IL-1β, IL-16, CCL11/Eotaxin, CCL13/MCP-4, CCL15/Leukotactin-1, CCL25/TECK, CCL26/Eotaxin-3, CCL27/CTACK, CXCL8/IL-8, CXCL10/IP-10, CXCL11/I-TAC, CXCL13/BCA, and  CXCL16/SCYB16. Hence, this study  has presented for the  first time the normal limits for a wide range of cytokines/chemokines in blood plasma  of the African inhabitants. Interpopulation differences were found, including those  for constitutive chemokines. Different levels of CCL19/ MIP-3β and CCL21/6Ckine chemokines (the CCR7 receptor ligands) for the two populations may indirectly indicate the physiological features of T-cell maturation. Increased levels of CXCR2 receptor ligands in the blood plasma  of Guineans, i.e.,  CXCL2/Gro-β, CXCL5/ENA-78 and  CXCL6/GCP-2, may be due  to additional function of these chemokines as ligands for atypical DARC chemokine receptor, which neutralizes chemokines from the blood flow, whereas 95% of West Africans have mutations in the DARC gene and do not express this receptor. Increased levels of proinflammatory IL-6  and TNFα cytokines, and chemokine CCL20/MIP-3α in blood plasma  from RG  residents may suggest inflammatory processes  in the liver, since 100% of the examined Guineans had antibodies against the hepatitis A virus, 48% had antibodies to hepatitis B virus (anti-HBs), and 12% had antibodies against hepatitis C virus. In summary, the differences in cytokine/chemokine level may be related  to specific environment, circulation of infectious diseases, composition of intestinal, skin and mucosal microbiota, as well as distinct genetic  features.

Heat  shock proteins (HSP, heat  shock proteins) form one of the cellular  molecular systems with chaperone activity, aimed  at stabilizing  the structure of intracellular proteins, ensuring  the resistance of cells to stress, renaturation of incorrectly folded  and  elimination of denatured intracellular proteins.Our task was to look for an association between  the presence of single nucleotide polymorphisms in selected  regions of the genome  and the basal level of transcriptional activity of the HSPA group genes, namely  HSPA1A/B, HSPA1A, HSPA1B, HSPA6  and  HSPA8  in mononuclear leukocytes  (PBMC), analyzed  in our  experiments volunteers from the population of the middle  part of Russia in order  to analyze  the universality of the biological  effects of these SNPs.The study was performed on DNA  and RNA isolated  from peripheral blood lymphocytes of 16 donors. Genotyping was performed by the polymerase chain reaction (PCR) followed by sequencing of the PCR product. To assess the level of gene expression  of the HSPA group, cDNA was synthesized on an RNA template isolated  from  PBMC cell fraction samples, followed  by real-time polymerase chain  reaction.The  following types of polymorphisms were genotyped: rs400547 (A/G), rs1150793 (G/A), rs707936 (A/G), rs707915 (A/T), rs376510 (T/C) located in the CLIC1,  MSH5, C6orf26, MSH5, C6orf25genes,  respectively. We determined the basal level of transcription of genes of constitutively expressed and inducible proteins of the HSP70 family and searched for the association of their  expression  with polymorphisms.It was found  that  in PBMC cells, DNA that has the following genotype: AG/AA (SNP rs400547), AG/GG (rs1150793), AG (rs707936), TArs707915, TC (rs376510), in the regions we studied, is associated with a decrease in the transcription of the HSPA1B  gene (p = 0.02) compared with homozygotes: GG (SNP rs400547), AA (rs1150793), GG (rs707936), TT (rs707915), CC  (rs376510).Associations of these  polymorphisms with  gene  expression  of HSPA1A, HSPA6  and  HSPA8 have  not  been  identified.The CLIC1,  MSH5, C6orf26, C6orf25 genes,  in which  the  polymorphisms studied by us are present, are located in the same locus near  the HSPA1B  gene on the 6th chromosome. We found  a decrease in HSPA1B  gene expression  in the presence of single nucleotide polymorphisms in nearby genes may indicate spatial interactions of this locus and the HSPA1B gene locus, and that a change in the genotype  CLIC1, MSH5, C6orf26, C6orf25 may entail a change in the expression  of closely arranged genes which are functionally significant  for the cell.

Lactobacilli are  widely used  in clinical  practice as probiotics, biologically  active  additives  and probiotic products for functional nutrition. Some  probiotics can  be considered as bacterial vaccines  due  to induction of immune response, accompanied by production of specific antibodies. The aim of the present study was to evaluate  the state of cellular and humoral immunity in women  by using probiotic strains of lactobacilli. The  study  included 31 healthy  women  aged  25-45  years.  As a source  of probiotic lactobacterial complex, we used  the  “Provag” preparation (RU  77.99.11.003.E.003746.02.11 of 11.02.2011, 1 capsule  contains 109 Lactobacillus  gasseri 57C,  Lactobacillus  fermentum  57A и Lactobacillus  plantarum  57B).  The  drug  was used for 30 days, at a rate  of one  capsule  per day. The  immune system was examined twice: before  administering the drug and after 30 days of treatment. The study of blood  lymphocyte populations and subpopulations was performed by flow cytometry using direct immunofluorescence technique. The concentration of IgA, IgM, IgG in blood serum was determined using enzyme  immunoassay. To determine specific antibodies, we used passive hemagglutination reaction with erythrocyte diagnosticum. The complex of probiotic lactobacilli Lactobacillus gasseri, Lactobacillus  fermentum  and  Lactobacillus  plantarum corresponding to the “Provag” preparation was used as a source  of antigen. It has been revealed  that  the number of T and B lymphocytes in peripheral blood increased after  30 days  of treatment with  the  probiotic preparation “Provag” in  healthy  women. Elevated contents of T cells was due to the T helper  cell fraction. Increased levels of T helpers and B lymphocytes were associated with stimulation of humoral immunity, as evidenced by increasing concentration of IgA and  IgG in blood  serum.  By means  of passive hemagglutination reaction, we have found  that  90% of healthy  women showed increased concentrations of specific IgA in blood after 30 days of treatment with “Provag” preparation.

The  purpose  of our  study  was to  examine the  effect  of immunomodulators (broncho-vaxom, immunovac-VP4 vaccine  and polyoxidonium) upon  the kinetics  of serum hydrolase inhibitors and lactoferrin in the treatment of community-acquired pneumonia (CAP). The  study included 71 CAP  patients at the age of 18 to 70 years. The  patients were divided  into  4 groups:  Group I (15 people)  was a control group  treated with basic antibacterial and  symptomatic therapy, according to the standard treatment regimen, without use of immunomodulators; the  patients from  group  II  (19  patients) were  additionally administered bronchovaxom (the  drug was prescribed upon  admission: 1 course  over 30 days, then  2 rounds  for 10 days each,  with an interval  in 20 days); group III  (20 cases) contained the patients who additionally received  polyoxidonium (the drug was prescribed from the 1st day of hospitalization, 6 mg daily i/m  for 3 days, then  10 injections over 10 days); group IV (17 cases): Immunovac-VP4 vaccine  was administered orally 4 ml and intranasally 2 drops on days 1, 4, 7, 10, 13, 19, 25, 31, along  with antibacterial and  symptomatic therapy. This  vaccine  consists of  antigens   from  opportunistic microorganisms (a  multicomponent  mixture of  water-soluble antigens   of S. aureus, K. pneumoniae, P. vulgaris, E. coli). Serum  concentrations of α2-macroglobulin and α1-antitrypsin hydrolase inhibitors were determined by the method of quantitative immunoelectrophoresis using the research test systems; lactoferrin (LF)  levels were evaluated by enzyme-linked immunosorbent assay using commercial test systems. These  indicators were studied  in blood serum  before treatment, on the 2nd, 13th, and 60th   days of observation.

It was shown that administration of immune modulators combined with antibiotic therapy  in patients with CAP can affect the kinetics of acute phase inflammatory proteins. The effect of broncho-vaxom corresponds to the classical pathway of the response to inflammatory process, i.e., activation of complex of positive acute phase reactants (α1-antitrypsin and  lactoferrin) and  inhibition (blocking) of negative  acute  phase  reactants (α2macroglobulin). Polyoxidonium has a noticeable effect only upon neutrophils secreting lactoferrin. ImmunovacVP4 promotes only short-term secretion of this protein. One may assume that activation of hydrolase inhibitors and lactoferrin after use of immunotropic drugs enhances clinical effect of therapy, with decreased severity and duration of symptoms, as well as lower exacerbation risk of chronic diseases and lesser volume  of drug intake. Antibacterial therapy  does not significantly  affect the kinetics of acute phase inflammation reactants in patients with CAP.  Administration of immunomodulators in combination with standard basic therapy  may affects the inflammatory process to different  degree,  thus, in turn,  leading to improved prognosis  in this disease.

Lifetime use of IgG replacement therapy  is the standard of CVID treatment. However, full control over stabilization of chronic infection loci is not always achieved, even if this therapy  is continuously applied. The purpose  of this study was to carry out comparative analysis of changes  in cellular  component of adaptive and  innate immune response, depending on effectiveness of replacement therapy  of patients with infectious CVID  phenotype. The  observation group  consisted of 15 patients with  CVID  who  were  diagnosed since early childhood in 100% of cases. They had prolonged respiratory infections followed by the development of complications requiring continuous treatment with antibiotics.

After  reaching mean  age of 15 years  old,  the  intensity of infection-associated antibody deficiency was 6-8  times  per year. After verification of the  diagnosis, the  patients received  replacement therapy, first at the saturation dose,  and,  after stabilization of IgG  at the level of 7-8 g/l,  at the monthly maintenance dose. The clinical  course  of the disease was traced  during  a full year of replacement therapy, and the cellular  immunity indices  were evaluated. In all patients, after a year of therapy  corresponding to clinical  guidelines, there  was an improvement in quality  of life indices, decreased rates of recurrent bacterial infections. At the same time, 40% of them continued to suffer, on average, 5.4±1.1 times a year and required long-term courses of antibiotic therapy. Evaluation of immune status did not reveal statistically significant  differences in IgG plasma saturation between the groups of patients with different treatment efficiency: 8.7 (8-9) g/l and 9.1 (8.5-10.5) g/l, at p = 0.5. The  differences related  to immune cell factors  in cases of smaller  effect of IVIG  therapy  are manifested in higher  relative  numbers of T effectors  containing lytic Granzyme B granules  and CD14⁺CD284⁺  monocytes, accompanied by lower spontaneous active  oxygen forms produced by neutrophils, lesser contents of CD16⁺ natural killers in peripheral blood.

The obtained data illustrate the value of monitoring, not only serum  IgG  level, but also the parameters of the  cellular  immune response. Such  analysis  may be essential  as a prognostic criterion for efficacy  of IVIG therapy. Reduced levels of some parameters of innate immunity cells serves a basis to formulate the concept of combined treatment and usage of tools that alter functions of immunocompetent cells.

A hybrid recombinant protein containing the amino  acid sequences of the three  most significant Pseudomonas aeruginosa antigens  (membrane proteins OprF, OprI  and toxoid  aTox)  was incorporated into a vaccine against Pseudomonas infection. Quality control of a hybrid recombinant protein and appropriate vaccine includes  determination of authentity and completeness of adsorption upon aluminum hydroxide adjuvant. The aim of our study was to develop  techniques of quality  control for a vaccine  based on the hybrid  OprF-aToxOprI  recombinant protein specific  to  P. aeruginosa.  Hybridomas secreting  specific  monoclonal antibodies for OprF-aTox-OprI were derived  from the fusion of myeloma cells and murine spleen  cells immunized with recombinant proteins P. aeruginosa. To  produce sufficient  quantities of antibodies, the  hybrid  cells were in vivo cultured in BALB/c mice.  Supernates and ascite liquids were chromatographically purified  with immune sorbent. Conjugation of antibodies with  horseradish peroxidase was carried  out  according to  P.K.Nakane. The  hybrid  OprF-aTox-OprI recombinant protein was detected by the  solid-phase ELISA, using a panel  of monoclonal antibodies and  conjugates of monoclonal antibodies with  horseradish peroxidase. Monoclonal antibodies were specific for different  OprF-aTox-OprI epitopes. Titration assays containing OprF-aTox-OprI protein at 78 ng/ml to 5000 ng/ml were used as quantitative standards for calibration curves.

To identify  the recombinant protein OprF-aTox-OprI, 55 variants  of of MAb pairs were tested.  Limits  of quantitative detection served  for selection of most  sensitive  and  specific  ELISA  variants.  The  quantitative detection limit was calculated for all 11 ELISA  variants.  Two ELISA  variants  with the highest  sensitivity were selected  for  quality  control of the  hybrid  recombinant protein. The  limits  of quantitative detection were, respectively, 2.9 and 13.6 ng/ml (0.0058  and 0.027% of the estimated antigen  content in the vaccine)  for the first and  second  ELISA  variants.  The  first variant  included a pair  of monoclonal antibodies specific  for the OprF  and OprI  epitopes, the second  variant  represented aTox and OprI  epitopes. Two variants  of ELISA  were developed to detect  the hybrid recombinant OprF-aTox-OprI protein. The first variant allows to determine the protein amount and to evaluate completeness of its adsorption on aluminum hydroxide. To confirm authenticity of the protein, both methods must be used, since they can detect all three antigens (OprF, aTox and OprI) which are present  in the fusion protein.

Tuberculosis is a widespread infectious disease caused by M. tuberculosis, which is one of the leading causes of death  in the world.  According to numerous literature data,  this is a genetically determined disease, and genetical polymorphism is a mechanism that leads to progression from infection to clinical  manifestation. Susceptibility to infection correlates with different genes at several loci, and each individual gene plays a unique role. It is known, that  the analysis of individual polymorphic variants  of genes does not provide  a sufficiently complete picture of the  mechanisms of formation of a predisposition to multifactorial pathologies, such  as tuberculosis, since  their  development is based  on complex intergenic and  gene-environmental interactions, which  must  be taken  into  account when  predicting the  risk of developing active  forms of the  disease  and  its severity. The concept of the functioning of cytokines as biomarkers of tuberculosis suggests that their products and interactions play an important role in the immunopathogenesis of the disease, because they form a cytokine chain  with  unique  functions, where  the  removal  of any  link in the  chain  disrupts  the  entire  mechanism of the  immuno-inflammatory process.  IL-6, together with  TNFα and  IL-1β, initiate early  pro-inflammatory reactions in  tuberculosis, stimulating local  and  systemic  inflammatory reactions under  participation of all common pro-inflammatory mechanisms with further  transition to activation of acquired immunity. Earlier, we carried  out a set of studies to evaluate  the association of alleles and genotypes  of these cytokine genes with a predisposition/resistance to pulmonary tuberculosis in Russians  of the  Chelyabinsk region.  These  studies have resulted  into assessment of certain distribution patterns of IL-1β, TNFα, IL-6  alleles and their genotypes in pulmonary tuberculosis and its various clinical  forms. The following methods were used: isolation of DNA samples  from whole blood,  genotyping of the studied  gene polymorphisms using PCR  and RFLP techniques. In this study, we analyzed  the intergenic interactions of the genes for the pro-inflammatory cytokines IL-1β, TNFα, IL-6 using the method of reducing multifactor dimension in patients with pulmonary tuberculosis. The program designs optimal models  of combinations for the studied  genes and their  interactions in tuberculosis patients. As a result of this study, a three-locus model  IL-6  (-174)*С – IL-1β (+3953)*Т – IL-1β (+3953)*С was established, which was characterized by 100% reproducibility and prediction accuracy of 72%. Among the analyzed  polymorphisms, the  IL-6  (-174)*C polymorphism possessed  the  highest  predictive potential with 15.27%.