Abstract. This review is focused on analysis of currently used flow cytometric methods designed for
identifying apoptotic cells in various invertebrate and vertebrate species. Apoptosis can be characterized by stage-specific morphological and biochemical changes that are typical to all kinds of eukaryotic cells. In this article, we consider different techniques of apoptosis detection based on assessment of cellular morphology and plasma membrane alterations, activation of intracellular enzymes and components of a caspase cascade, as well as DNA fragmentation and failure of mitochondrial transmembrane potential, as assessed in various animal groups. Apoptosis recognized as a key mechanism aiming at maintenance of cellular homeostasis in multicellular organisms, and such investigations represent a necessary component of fundamental and applied studies in diverse fields of experimental biology and immunology. A broad spectrum of apoptosis markers is
used, and the preference is given to optimal approaches, as determined by experimental tasks, and technical opportunities of the laboratory.

Abstract. This study has shown that that a common cytokine pool induced in cultured human peripheral blood cells (PBC) supplied by either γ-globulin fraction proteins, copper or zinc cations, or appropriate metal complexes, contains detectable amounts of IL-6 (0.39+0.14 to 2.04+0.16 ng/ml). γ-globulin complexes with zinc or copper ions are able to induce production of IL-6 in amounts differing from those induced by control proteins, or copper and zinc ions used alone. IL-6 production by PBC in presence of γ-globulin/zinc complexes was 1.5 to 2.5-fold higher than with control proteins or single zinc ions at 24, 48 and 72 hrs of observation, whereas IL-6 production by PBC in presence of γ-globulin complexes with copper was higher than with appropriate control proteins or copper ions used alone for 48 and 72 hrs of cell incubation (resp., 1.7 to 2.4 and 2.8 to 4.6-fold increase). Possible role of copper and zinc ions chelated by γ-globulins from the microenvironment and modifying the Fc regions of antibodies, is considered a potential regulatory factor of IL-6 production by human PBC.

Abstract. The article includes results of studying frequency distributions of polymorphic genotypes for
some genes regulating angiogenesis, i.e., MMP and VEGF, and their combinations with genotypes of some cytokines showing pro-angiogenic activity in patients with diabetes mellitus type 2 (DM2) and healthy controls. Angiogenesis-inducing MMP2-MMP9-VEGF genotype constellations have been shown to be more common in DM patients, thus presumably causing an imbalance between activation and suppression signals, being connected with C alleles of VEGF and MMP2, as well as with harboring of MMP9 C allele. Certain genotypes of IL-1B, IL-4, IL-10, IL-6, and TNFA, along with MMP and VEGF variants are more common within combined genetic patterns in DM patients. It is suggested that allelic combination studies of angiogenesis- and inflammation-regulating genes are necessary for understanding the DM2 pathogenesis, taking into account genetic control mechanisms influencing basic production levels of these regulatory factors.

Abstract. We studied cytokine profile in blood and exhaled breath condensate (EBC) in patients with chronic obstructive pulmonary disease (COPD) being in remission state. It is shown that pro- and anti-inflammatory cytokine contents depended on the disease severity, both in whole blood and EBC of the COPD patients. We have revealed an increase in TNFα, s-TNFα RI, TGF-β1 and bFGF in EBC of patients with COPD manifestations, thus being indicative for progression of metabolic changes in lung tissue, and advanced stage of respiratory functional disturbances. Cytokine profile abnormalities in COPD patients resulting, in part, from systemic and local disorders of cellular immunity, represent a major pathogenetic mechanism determining the disease progression.

Abstract. Over recent years of studies in infantile cerebral paralysis (ICP), the changes in periventricular area are given great importance, especially, periventricular leukomalacia. An immunopathological process may define the prenatal brain damage. We performed a clinicalytogenetic and immunological evaluation, including subpopulation analysis of lymphocytes, functional activity of phagocytes, quantitative content of TNFα in blood serum and saliva in thirty-five ICP patients (1 to 4 years old). This study has shown suppression of cellular immunity, increase in oxidation/reduction activity of neutrophils, and increased TNFα levels in peripheral blood serum and salivary fluid in cases of periventricular leukomalacia that resulted into ICP. An interrelation was revealed between the levels of genome instability and TNFα content, thus suggesting involvement of active immunopathological processes in those patients with ICP who develops periventricular leukomalacia.

Abstract. We have performed immunological studies in forty-five patients who underwent lamellar keratoplasty by means of Alloplant biomaterial. Serum antibodies to eye tissue-specific antigens (bovine corneal protein (BCP)-54, lens alpha-crystallin, S-antigen) were tested, and IgG, A, M in the lacrimal fluid were also determined, to detect cellular sensitization against same antigens, and to evaluate the levels of local non-specific inflammation. Percentage of cells activated for BCP-54 was found to be significantly decreased after surgery (p < 0.04), followed by increase to initial levels (p > 0.20) at later terms. Percentage of cells activated for lens α-crystallin was shown to be decreased upon further observation (p < 0.04). A post-surgical increase in IgG levels was revealed in lacrimal fluid (p < 0.02), followed by a decrease later on (p < 0.002). Thus, application of Alloplant biomaterial for lamellar keratoplasty does not cause any sufficient tension of specific humoral immunity against ocular tissues, as evidenced by lack of statistically significant changes of humoral and cellular immunity during post-surgical observations.

Abstract. Autologous hematopoietic stem cell transplantation (auto-HSCT) is one of the most effective methods for treatment of patients with various forms of hemoblastoses, both in adults and children. However, high-dose chemotherapy protocols used in this procedure are characterized by pronounced myeloand immunotoxicity. Appropriate data concerning immune state at long terms after high-dose chemotherapy and auto-HSCT are sparse and controversial, and there is no consensus on time dynamics of immune system reconstitution. The aim of this study was a comprehensive evaluation of immunity in recipients of auto-HSCT at longer terms. Clinical and immunological testing was performed in ninety-eight patients with hematological malignancies before starting a high-dose chemotherapy, and at late post-transplant period. The state of cellular immunity was assessed as expression of surface CD3+, CD4+, CD8+, CD16+, CD19+ lymphocyte antigens. Humoral immunity was evaluated by serum IgG, IgA, and IgM levels. The studies have revealed disorders of cellular and humoral immunity, as well as nonspecific immune resistance factors in recipients of autologous hematopoietic stem cells at late terms post-transplant. Immune reconstitution in patients receiving highdose consolidation treatment followed by auto-HSCT takes longer time than in patients who did not receive autologous hematopoietic stem cells. Severity of these disturbances and immune reconstitution rates depend on the type of conditioning regimen, and the source of haematopoietic stem cells used for transplantation.

Abstract. We studied immune resistance efficiency of vaccination against influenza A/California/7/2009(H1N1)v in women at second trimester of physiological pregnancy in a blind, placebocontrolled study. The first group included thirty pregnant women who were injected by univalent subunit “MonoGrippol plus” vaccine. The second group consisted of thirty-seven pregnant women immunized by trivalent “Grippol plus” vaccine. Thirty-one pregnant women (III group) received placebo treatment. Non-pregnant women (IV group) were injected with “MonoGrippol plus”. We did not find any differences in clinical features of vacccine-challenged time period in pregnant women from groups I-III. Notably, sufficient numbers of women were found to be seroprotected 1 month post-vaccination (I group, 80.0% ; II group, 75.7% ; IVgroup, 80.6% ) with high levels seroconversion (I group, 46.6%; II group, 51.4%; IV group, 53.3%). Within 9-10 months after vaccination, a decreased seroprotection was revealed in II group of pregnant women. More stable specific immunity levels were detected for the groups immunized with univalent vaccine.

Hence, the local subunit adjuvant “MonoGrippol plus” and “Grippol plus” vaccines were shown to exibit a high immune resistance efficiency profile and clinical safety, when used in pregnant women, thus presuming an extended application field for these biological drugs in public health service. State of pregnancy seems not to be a limiting condition for induction of specific immune resistance.

Abstract. Present study dealt with efficiency of melatonin implementation in a combination therapeutic schedule of bronchial asthma (BA). A group of 248 patients with atopic, or mixed clinical forms of BA being in exacerbation, or medication remission state, and 36 healthy donors were included into the study. Melatonin (Melaxen, Unifarm, USA) was administered as a single daily dose of 0.003 g, at 21.00, accompanied by a standard therapy in twenty BA patients for 21 days. We determined contents and functional properties of Т- and B-lymphocytes, mononuclear phagocytes, IgE, IL-4, IFNγ levels, as well as melatonin concentrations in blood serum in the morning and evening time. When included into BA treatment protocol, melatonin proved to cause partial restoration of circadian rhythm for Tand B cell subpopulations, mononuclear phagocytes, cytokine production, due to its chronotropic and immunomodulating activity. This effect is associated with a more pronounced clinical effect, thus presuming reversibility of desynchronosis state.

Abstract. Some molecular biomarkers of peripheral blood mononuclear cells (PBMC) were studied in patients 18 to 30 years old with community-acquired pneumonia (n = 30) and healthy persons (n = 15), along with testing appropriate biological effects of low-intensity microwave irradiation (1000 MHz), at a dose rate of 100 pW/cm2. Concentrations of cytokines and some molecules providing intracellular signal transduction in PBMC were measured by ELISA tests. Biological action of microwave radiation was studied after 1-hour irradiation of whole blood, followed by incubation for 24 hours. A single round of irradiation was shown to cause an increase in NFκB content by 12.5% (р = 0.001); IκB by 21.1% (р = 0.0007); phosphorylated JNK1/2, by 18.2% (р = 0.052); p21 protein amounts, by 56.2% (р = 0.031); IL-2, by 8.5% (р = 0.08); IL-4, by 17.6% (р = 0.031). Antioxidant potential of PBMC supernate
was augmented by 65.2% (р < 0,001). Hence, a single 1000-MHz microwave irradiation modulates activity of intracellular events in PBMCs from pneumonia patients.

C1q- and C3d-IC, were determined in patients with posttraumatic stress disorder (PTSD), as compared to healthy subjects, using an enzyme-linked immunosorbent assay. According to the data obtained, the levels of both C1q- и C3d-IC were significantly increased in PTSD-patients versus healthy subjects, thus reflecting hyperactivation of classical complement pathway detected in this pathology. Moreover, a significant positive correlation was found between C1q- and C3d-IC levels in PTSD group. It was concluded that PTSD is characterized by altered mechanisms of IC recognition and elimination that may underly chronic activation of immune system and systemic inflammatory response associated with this disorder.

Abstract. Immunological characteristics of phagocytic activity and cellular immunity are presented for 112 patients with acute destructive pancreatitis with suppurative complications (group I), versus non-septic cases. We evaluated lymphocyte subpopulation profiles, phagocytic activity of leukocytes, serum levels of interferon-γ and circulating immune complexes. A mixed-type secondary immune deficiency was shown to develop in pancreatic necrosis. Distinct signs typical to immune deficiency, as well as lack of cellular immune potential were evident as soon as by day 3 of the disease.

Abstract. The aim of present study was to investigate some indexes of immune deficiency, antimicrobial protective mechanisms, and lipid-releasing ability of leukocytes in patients with community-acquired pneumonia. The study groups involved fifty-two pneumonia patients and 26 healthy people. The patients were subdivided into two age groups, 20-40, and 41-60 years old. We classified the laboratory findings according to severity and clinical phase of the disease, and smoking status. Evaluation of immune deficiency included filling of special questionnaire, drawing of scores, and stratification into two groups, according to presence or absence of clinical immune deficiency. Serum concentrations of IL-6, IL-8, TNFαα and CRP, were detected by enzyme immune assay. An original technique was applied for detection of lipid-releasing ability of leukocytes. It was shown that 46 per cent of pneumonia patients showed clinical signs of immune insufficiency, being more common in cases of severe pneumonia, or extensive lung damage, according to radiological data. A relative quantitative deficiency of serum IL-8 and TNFαα was revealed in patients with severe pneumonia, accompanied by elevated serum IL-6. Lipid-releasing ability of leukocytes was significantly decreased among patients between 20 to 40 years old. This deficiency was more expressed in patients with severe pneumonia, and it returned to near- normal levels upon clinical resolution of the disease.

Abstract. Twenty patients with basal cell skin carcinoma at the age of 56 to 70 years have been included into study. We have investigated multiple parameters of cellular and humoral immunity, features of phagocytic activity, spontaneous and stimulated chemiluminescence of peripheral blood neutrophils. Distinct changes in blood lymphocyte subsets have been assessed by immunophenotyping. Changes in serum immunoglobulin profiles and increased levels of circulating immune complexes were revealed. Altered phagocytic activity and oxygen-dependent metabolism of blood neutrophils were also detected.