Abstract. Flow cytometry is considered as a modern technology for fast measurements of cellular characteristics, their organelles, and processes occurring within them. It is regarded as an efficient solution in many important areas of cell biology, immunology and cellular engineering. Present article bears on main developments in flow cytometry and their applications in medical and biological practice. Usage of modern achievements in fluorescent dyes, progress in laser and computer technologies, as well as potent software, resulted in wide application of this technique in medical practice. Accordingly, usage of monoclonal antibodies conjugated to different fluorochromes has led to elaboration of multiparametric analysis and did sufficiently simplify specialized works aimed for diagnostics of various immune disorders. The new directions in flow cytometry, e.g., flow cytoenzymology, provide wide opportunities for detailed identification of damaged or altered cells, and taking adequate decisions in treatment of detected pathological changes. The authors suggest that this article could initiate a series of publications concerning usage of this technology and its modern applications in broad laboratory practice.

Abstract. Chronic hepatitis C (CHC) is widely spread disease with a long incubation period and weakle expressed clinical features. Pathogenesis of CHC despite of intensive efforts is still unclear. Due to scarcity of clinical signs diagnosis and monitoring of CHC represent considerable difficulties. Chemokines and chemokine receptors are fascinating object for investigation since they are mediators of chronic inflammation. Therefore, studying local and systemic immune response by evaluating expression of genes encoding chemokines and their receptors is of sufficient interest. In present study we investigated peripheral leukocytes samples and liver biopsies from 21 CHC patients. Blood leukocytes of 10 healthy volunteers were studied as a control for expression of mRNA for chemokines and their receptors. For all biopsies histological examination including evaluation of fibrosis and histological activity index was performed. mRNA’s encoding MIP-1α, MIP-1β, MCP-1, RANTES, CCR1, CCR2, CCR3, and CCR5 were under study. We found that the level of liver MCP-1 mRNA expression is increased reflecting degree of disease progression, moreover it was significantly higher in liver as compared with blood. The results of study showed that the changes in chemokine/receptor system in blood of CHC patients were significantly stronger as compared with intrahepatical expression of specific mRNA’s. The contents of CCR1, CCR2, CCR3, and CCR5 mRNA in patients with minimal histological manifestations were significantly lower than in group with more severe ones. CCR1 and CCR5 mRNA expression did directly correlate with histological activity index and fibrosis degree. The values MCP-1 mRNA’s in liver biopsies were in direct correlation with histological manifestations. In general, our study shows that CHC progression may be connected with local activation of MCP-1 gene thus leading to systemic response as increased expression of CCR1, CCR2, CCR3, and ССR5 genes in peripheral blood leukocytes.

Abstract. We performed measurements of serum interleukin-6 (IL-6), interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), as well as total contents of α2-macroglobulin (MG) in blood and concentration of its active transport form, i.e., circulating complexes with plasmin (MG-PL) in women at their reproductive age, suffering with tubal infertility, who participated in an in vitro fertilization (IVF) program. We have found that the cohort of women with tubal infertility is initially heterogeneous and consists of persons with relative enhancement of either humoral, or cellular immune response. Stimulation of superovulation upon IVF in the women with increased humoral immune reactivity promotes normalization of cytokine levels, increases total contents of MG and maintains its active form (MG-PL), both by transporting bioactive substances, which are necessary to growth of the embryo, favors better access of MG-PL to cells due to reduction of IFNγ levels. Finally, it brings about positive results of the IVF procedure. In women with increased cellulartype immune reactivity, the MG levels are initially low, along with high IFNγ levels, thus diminishing transport of biologically active substances to the cells by MG-PL, as well as high and non-decreasing TNFα concentrations after stimulated superovulation, and, probably, an increased activity of NK-cells and cytotoxic lymphocytes. These factors minimize the possibility of favorable IVF outcome. With respect of the trends revealed, we would like to recommend determinations of serum cytokines and MG-PL complexes, as an additional prognostic tool when carrying out IVF programs.

Abstract. Comparative study was carried out, aiming to assess immunostimulatory properties of high-molecular chitosan (Ch-HM) and its derivatives, i.e., low molecular weight chitosan (Ch-LM), N-3-hydroxymyristoyl (Ch-LM) at a low acylation ratio, N-3-hydroxymyristoylchitooligosaccharides (N-acylchito-biose, -triose, -tetraose), N-, O-carboxyalkylchitosans (carboxymethyl, -ethyl, -propyl derivates). It was established, that the chemical modifications of chitosan influenced its biological activity. The derivatives of chitosan were found to have improved physical properties (good solubility in neutral and alkaline solutions, low viscosity in acidic solutions, good absorption from a gastrointestinal compartment), as compared with initial (high molecular weight) chitosan formula. They possess immunomodulatory properties and may be regarded as promising substances for preparation of medical drugs and biologically active food additives (BAFA).

Abstract. The aim of present study was to determine the features of STAT6 and phospho-STAT6 (pSTAT6) expression in bronchial asthma (BA). Patients and methods. Eleven patients with allergic (atopic) steroidfree were examined, five healthy controls served as a control. Expression of proteins (STAT6 and pSTAT6) in peripheral blood lymphocytes was studied by Western blot analysis after cell lysis. Preparation of cell lysates and Western blotting were performed using a standard procedure (Amersham). Antibodies against pSTAT6 and STAT6 (manufactured by Cell Signaling) were used. Relative levels of specific proteins were analyzed using actin as a reference, by means of anti-actin antibody. Results. STAT6 phosphorylation was significantly increased in lymphocytes of patients with BA exacerbation, as compared to patients in remission and healthy group. The level of STAT6 was significantly higher compared to healthy persons and showed negative correlation with grade of air flow obstruction. Conclusion. STAT6 and their active form pSTAT6 may play a key role in BA pathophysiology. This study suggests atopic, steroid-free BA (in particular, on exacerbation) to be associated with active cellular inflammatory process, involving activation of STAT6, along with increased level of their active form (pSTAT6). The work was supported by Saint-Petersburg government grants: PD04-4.0-102 (Certificate N ASP604079).

Abstract. Malignant diseases of blood system, associated with an eosinophilic syndrome, are accompanied by altered production of some key cytokines regulating proliferation, differentiation and activation events in the cells of eosinophilic lineage, as detected with ELISA approach. An imbalanced secretion of Th2 cytokines, as studied in lymphoproliferative diseases, may be a promoting factor of eosinophilia development in malignant disorders of blood system.

Abstract. Some immune parameters (cell subsets in peripheral blood, spontaneous and LPS-induce production of IL-1β, IL-5, IL-7, IL-12, IL-13, IL-17, G-CSF, MCP-1 and MIP-1β in whole blood cultures) were studied in fifty patients with breast cancer (T2-3, N0-3, M0) after radiation therapy and surgical treatment. A significant decrease in lymphocytes, CD3+ and CD4+T-cell counts, CD19+B-lymphocytes, erythrocytes, hemoglobin level, and amounts of phagocytic cells was revealed. The cytokine status was characterized by prevalence of pro-inflammatory cytokines (IL-1β, IL-12, IL-17) and chemokines (MCP-1, MIP-1β), accompanied by low levels of Th2/ anti-inflammatory IL-13. Furthermore, a markedly increased production of G-CSF and IL-7 was found, thus apparently pointing to the switching of a compensatory mechanisms in response to cytoreductive effects of anti-tumor therapy. IL-7 levels in lymphopenic patients (< 1.2 х 109/L; a mean of 0.81±0.11) were significantly higher than that in an opposite group of lymphopenia-free women (> 1.2 х 109/L; a mean of 1.34±0.01). An inverse correlation (rS = –0,88; p < 0,0001) between blood lymphocyte counts and IL-7 levels allows us to suggest a mechanism of homeostatic peripheral expansion (HPE) to be involved in maintenance and restoration ofT cell homeostasis in the patients treated for breast cancer. Significance of HPE mechanism for induction of both beneficial protective tumor-specific autoimmunity and increased risk of autoimmune complications is discussed.

Abstract. Taking into account close localization of genes encoding HSP70-2 and TNFα and mutual influence of their products upon development of septic process, we investigated allelic polymorphism in the coding region of HSP70-2 gene at position 1267, and a promoter polymorphism of TNFα gene (–308 position) in sixty-one patients with surgical sepsis, as compared with 100 healthy donors, assuming that certain genomic variants can be associated with susceptibility for sepsis and/or fatal outcomes in severe sepsis. Significant differences in frequencies of HSP70-2 gene alleles have been found in the group with sepsis against healthy controls. Prevalence of HSP70-2*B/B allele is increased in the group of patients with sepsis, and a predisposal for sepsis development is shown for carriers of the given genotype. When analyzing carriers of combined HSP70-2/TNFα allelic variants among dead patients vs survivors, we have revealed specific combinations that are detected solely among non-survived patients, i.e., HSP70-2 A/TNFα AA, or only among survivors (HSP70-2BB /TNFα AA и HSP70-2 BB/TNFα AG). The results obtained allow us to suggest about certain genetic factors that may sufficiently influence intensity of inflammation in sepsis and, hence, upon clinical manifestations and outcomes of the disease.

Abstract. The study was aimed for evaluation of clinical and immunological efficiency of an immunomodulating drug Licopid (Peptec «CJSC») in chronic abacterial prostatitis (CAP) accompanied by fertility alterations. Licopid contains N-acetylglucoseaminil-β1-4-acetylmuramoyl-L-alanyl-D-isoglutamine (GMDP) which mimics a minimal repeated peptidoglycan structural unit from bacterial cell wall, and it represents a pathogen-associated molecular pattern, able to influence the innate immune system via appropriate pattern-recognition receptors. Thirty-eight infertile patients with azoospermia were under observation (20 to 45 years old, a mean of 28.84±1.1). CAP was diagnosed in accordance with NIH criteria, as chronic prostatitis of III-A category. Clinical examination included sperm analysis, using WHO criteria, and phenotyping of peripheral blood mononuclear cells, by the means of standard flow cytometry (FACScan, «Becton Dickinson»). A control group consisted of eight fertile males 21 to 33 years old (a mean of 26.8±2.33) with normozoospermia. For twenty-four randomly selected patients (main group), a treatment with Licopid was applied (a total of 100 mg, at a daily oral dose of 10 mg), in addition to common therapeutic schedule (fluorochinolones, α-adrenoblockers). A comparison group included fourteen patients receiving common therapy. Some differences in immune response were detectable in the CAP patients. Along with balanced («intense») state of immunity revealed in 16 subjects, 22 persons did exhibit a systemic immunosuppression which manifested as alterations in MNC activation and decreasing efficiency of the standard treatment. Licopid seems to be a drug of choice for these cases, due to decreased CAP activity and normalization of a spermogram following its application. Differentiated administration of immune modulating drugs will allow prevention of adverse effects of empirical immunocorrection.

Abstract. We examined immunological state in 100 patients with acute non-lymphoblastic leukemia (ANLL, M4), and in 73 cases of acute lymphoblastic leukemia (ALL, common variant). The most expressed disturbances of immune status in ANLL patients were revealed during relapses, and in patients with ALL they were detected in the state of clinical remission. In cases of ANLL, T cell immunodeficiency did develop in crisis and remission, whereas combined T- and B-cell immunodeficiency occurs during relapse of ANLL. T cell immunodeficiency was found to develop at all stages of ALL. These changes and differences may characterize specific immunopathogenetic aspects of development and progression of ANLL and acute ALL.

Abstract. Cytomegalovirus (CMV) infection in diploid human fibroblasts (HF) and levels of cell resistance to this virus were shown to be in direct correlation with high α-interferon (IFNα) gene activity and induction of IFNγ gene transcription. Regulation of IFNα mRNA transcription was revealed to be positively associated with cellular DNA synthesis. At the same time, activities of IFNβ and IFNγ genes were at the constantly low level and were not induced in DNA-synthetic phase (S-phase) of the cells. Levels of IFNα mRNA synthesis are quite different for G0- vs S-phase-synchronized HF110044 cell cultures: appropriate values for dividing cells (S-phase) proved to be 100-fold higher than in resting state (G0). The mode of CMV infection in resting HF-cell could be considered either as acute, or a productive one. On the contrary, proliferating cells exhibited lagging viral syntheses and delayed cell death. Arrest of CMV replication may be, to some extent, comparable with latent infectious state, being associated with high production of IFNα. Both basal and induced levels of IFNα mRNA in CMV-resistant adult human skin fibroblast cells (HSF-1608) were 10-fold higher than in human embryo lung cell line (HELF-977), which is highly sensitive to CMV. Moreover, a short-time induction of IFNγ genes was observed in resistant cells, whereas no such effect was noticed in highly sensitive cells. CMV reproduction in sensitive cell lines (HELF-977 and HELF-110044) partially inhibits IFNα mRNA transcription at the later stages of infection (24 to 48 hours). Thus, cellular resistance and control of CMV infection in diploid fibroblasts are associated predominantly with high transcription of IFNα gene, and with temporal induction of IFNγ gene. We did not reveal any participation of IFNβ genes in protection of human diploid fibroblasts from CMV.

Abstract. Eosinophils are regarded as major cell population directly involved into late phase of allergic inflammation of nasal mucosa. We performed a comparative study, measuring the amounts of eosinophils and eosinophilic cationic protein (ECP) in nasal secretions and serum samples of thirty-three patients with persistent allergic rhinitis (PAR), and twenty-five persons with chronic infectious rhinitis (CIR), to determine diagnostic significance of these parameters. Eosinophil counts in nasal secret were carried out by exfoliative cytology analysis. Sampling of nasal secretions was performed by absorption method. The samples were standardized by concentration of urea in nasal secretions and sera. ЕСР levels in nasal secretions and serum were determined by chemiluminescence assays (UniCAP 100, Phadia, Sweden). Mean scores of eosinophils in nasal secret of PAR patients were 4.6 times higher, than in the patients with CIR. Median ECP values in serum and nasal secretions of PAR patients were, resp., 30.5±28.0 μg/l and 402.7±326.9 μg/l. In patients with CIR, ECP values were 12.4±11.5 μg/l in serum and 86.9±137.4 μg/l in nasal secretions. The appropriate differences have been significant (р < 0.05). No correlations were found between ECP levels in serum and nasal secretions, as well as between ECP levels and eosinophil counts in nasal secretions. The technique of ECP determination in nasal secretions was the most sensitive approach, whereas counting of eosinophils proved to be the most specific method. ECP levels in nasal secretions well characterize an active phase of allergic inflammation and may be used as quantitative parameter for estimation of PAR severity.

Abstract. In present article, essentially new methods are described for evaluation of adhesion of peripheral blood mononuclears to human endothelial cell line Ea.Hy926. These methods are suitable for both experimental purposes and research in clinical immunology, including laboratory diagnostics of immune deficiencies, infectious diseases, complicated pregnancy, and other kinds of clinical disorders.

Abstract. The article considers an original approach to individual dose selection and efficiency evaluation of immunity-targeted drugs applied for treatment of children with trauma, based on examination of phagocyte locomotive functions.

Abstract. A study of leukocyte regulation system in patients with exacerbated bronchial asthma (BA) was performed by means of measuring spontaneous and induced production of certain cytokines (IL-4, IL-1b, IL-10 and IFNg) in whole blood, and drawing some specific coefficients reflecting production of the cytokines, as compared to control values. Group 1 consisted of mixed-type BA patients, with mild clinical manifestations at exacerbation stage. A control group included twelve healthy persons. The cytokine network was evaluated according to spontaneous production of cytokines (IL-4, IL-1b, IL-10 and IFNg), or their induction with standard mitogens (PHA and E. coli LPS). The results obtained have shown that in BA as a clinical model of chronic inflammation, synthesis and activation of the cytokines depend on the phase of disease. At stage of exacerbation, an imbalance occurs between pro- and anti-inflammatory cytokines, with predominance of anti-inflammatory IL-10, which is targeted to suppression of bronchial inflammation.

Abstract. Participation of pro- and anti-inflammatory cytokines in pathogenesis of inflammation and fibrosis served as a basis for studying TNFα and TGFβ levels in blood serum from the patients with chronic hepatitis C (CHC), as compared with modes and degree of patho-morphological changes in liver biopsies. We have detected a sufficient increase in serum TGFβ levels from patients in sub-clinical stage of CHC, independent on activity of the inflammatory process and expression of fibrosis, with a trend for its higher levels in cases of clinical decompensation at cirrhotic stage of the disease. Ambiguous changes of TNFα indexes in CHC patients with a tendency of higher levels in cases of higher histological activity and fibrosis of liver tissue allow us to consider a possible correlation with varying degree of compensatory and adaptive reactions.

Abstract. Statins are widely used to lower lipid levels and prevent cardiovascular disease. Recently described anti-inflammatory and immunomodulating properties of statins substantiate their application as a potentially novel treatment for autoimmune diseases. We evaluated efficacy and safety of simvastatin (40 mg daily) in patients with active rheumatoid arthritis (RA), as a supplement to stable doses of disease-modifying antirheumatic drugs. After 12 weeks of treatment, there was registered a significant decrease in disease activity score (by 0.89 points), improvement of life quality, and attenuation of pain levels. To confirm efficacy and safety of simvastatin in active RA, a large-scale controlled randomized study should be performed.

Abstract. We studied some effects of a biologically active food additive (BAFA) «DNAaC» which contains DNA from salmon soft roe and vitamin C as active substances. This BAFA was investigated as a complementary remedy during radiation therapy of women with breast cancer (BrC), with respect to dynamics and levels blood serum cytokines, i.e., early hematopoietic factors (IL-3, GM-CSF, TNFa), and balance of marker cytokines for Th1- and Th2-response (IFNγ, IL-10). It was shown that «DNAaC» has modulating effect on secretion of all investigated cytokines. There was, however, a higher and statistically valid corrective effect upon GM-CSF, along with increase of initially low IFNγ and TNFa concentrations, and reduction of initially high IL-10 levels. Hence, «DNAaC» contributes to improvement of cell-mediated immune deficiency, stabilization of hematopoietic system, and brings about tolerance to basic treatment approaches in oncological diseases.

Abstract. In vitro response of blood mononuclear cells to cancer-associated antigens was studied in women with genital pathology, and these results were compared with serum levels of pro- and anti-inflammatory factors of immunity, like as with histological features of genital cancer and cervical epithelial dysplasia. In vitro cellular response was regarded as positive, if relative amounts of CD8-positive lymphocytes increased by > 15% following incubation of blood mononuclears with cancer-associated antigens. Positive reaction and elevated serum levels of anti-TNFα and anti-IFNγ antibodies were associated with lesser malignancy of tumor, as proven by histological findings in the women with genital cancer. A positive cellular reaction was associated with increased serum levels of IFNγ and anti-TNFα in women with grade II–III cervical epithelial dysplasia. It is concluded about potential applicability of testing mononuclears with fetal proteins, to determine a grade of malignancy for the female genital tumors, as well as a degree of regenerative disturbances of cervical epithelium.

Abstract. We have observed 128 patients with non-nosocomial pneumonias. Bacterial microflora was isolated in 22.6 per cent of entire group. 17 per cent of the samples were seropositive for Mycoplasma pneumoniae, along with detectable DNA specific for this causal agent. In 25 per cent of the patients, isolation of bacterial microflora was associated with positive antibody titers to Mycoplasma pneumoniae. Serum contents of cytokines (TNFα, IL-8, IL-6) have been studied in the patients. A positive correlation was found between increased cytokine levels and severity of the disease.

Abstract. Levels of C-reactive protein and immunoglobulin G antibody titers to H. pylori in blood at patients of ischemic heart disease were measured, dependent on clinical course of disease. It was revealed that more expressed acute-phase changes in blood (leukocytosis, increased C-reactive protein contents) in the patients with unstable angina and acute myocardial infarction, as compared with appropriate parameters in stable stenocardia, were accompanied by increased titers of IgG antibodies against H. pylori.

Abstract. The aim of present work was to evaluate clinical significance of B-lymphocytes spontaneous antibody-synthesizing activity by B-lymphocytes (LASA) in patients with rheumatic inflammatory diseases (RD), i.e., reactive arthritis (ReA), ankylosing spondylitis (AS), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). Significantly higher LASA levels were revealed in the patients with ReA, AS, RA, and SLE, as compared with healthy persons and patients with osteoarthrosis. Clinical significance of LASA indexes and their changes may reflect manifestation and degree of immunological activities in ReA, AS, RA, and SLE.

Abstract. In HIV-infected patients, the process of antibody production to certain HIV-I structural proteins proceeds in differential manner, depending on the antigen localization. Upon progression of the disease, an increased ratio of antibodies to env surface glycoproteins is found, along with decreased percentage of antibodies to gag gene proteins.

We have studied distribution of IL-1β gene allelic variants (-511C>Т polymorphism in promoter region and IL-1β +3953C>Т polymorphism in exon 5) in sixty-two patients with recurrent and protracted rhinosinusitis, as well as their intercorrelations between allele frequencies, IL-1β levels and predisposal for reccurence of chronic rhinosinusitis (CR). DNA samples were genotyped using PCR-RFLP analysis. IL-1β production by PHA-stimulated lymphocytes were determined in the cell supernates by means of quantitative enzyme immunoassay. In majority of patients (> 70%) with CR, decreased production of IL-1β was found, irrespectively of disease recurrence. Frequency of a «low-producer» homozygous *С/*С genotype +3953C>Т of IL-1β gene was two-fold higher in the patients with CR than in healthy controls (OR=3.4). In patients with protracted, recurrence-free patients, this parameter showed only a tendency to increase. Decreased production of IL-1β was associated with homozygous *С/*С genotype +3953C>Т of IL-1β gene (OR = 3,4), whereas high levels correlated with heterozygous С/*T genotype (OR = 7,6).

Thus, low IL-1β production in carriers of homozygous *С/*С of +3953 polymorphic locus of IL-1β gene represents a diagnostic marker reflecting a risk of recurrent disease. Hence, a pathogenesis-based therapy of purulent rhinosinusitis with protracted and recurrent clinical course is possible by means of recombinant IL-1β (e.g., Betaleukin, St.Petersburg Institute for Highly Pure Biological Preparations), being a highly effective approach which favors decreased recurrence of disorder.

Abstract. Sixty-nine children (5 to 14 years old) with infectious mononucleosis caused by EBV were observed in acute phase and during recovery period. The indexes under study did evaluate cell-mediated and humoral immunity, levels of spontaneous and mitogen-induced production of IL-2, IL-10, IFNγ by peripheral blood mononuclears. We have founded appropriate changes to be dependent on clinical phase of the disease. The most significant changes were revealed during acute phase of disease. A positive dynamics of cell-mediated and humoral immune response was observed during convalescence period. However, complete normalization of most immunologic indexes and cytokine status did not occur, corresponding to the clinical signs. This situation necessitates immune rehabilitation to be carried out for the children having at this phase of infectious mononucleosis.