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Macrophages are involved in the regulation of fibrogenesis and turnover of the extracellular matrix. One way to perform this function is through the production of profibrotic and fibrolytic factors including fibronectin, laminin, collagen, and extracellular matrix proteases. The production of most of them has been well studied in experimental models; however, much remains unclear regarding human macrophages. Therefore, the aim of this study was to study the content of extracellular matrix proteases (MMP-2 and MMP-9, cathepsin L), their inhibitors (TIMP-1), and collagen (type I) in supernatants of differently activated human macrophages. We compared macrophages differentiated by M-CSF or GM-CSF and further polarized in M1 with lipopolysaccharide, in M2a with IL-4, and in M2c with dexamethasone. Macrophages was obtained from peripheral blood monocytes. The content of MMPs, TIMP, cathepsin, and collagen was determined using appropriate ELISA kits. The results obtained demonstrate that differentiation factors are more important for the production of the above factors compared to polarizing stimuli (lipopolysaccharide, IL-4, dexamethasone). Moreover, macrophages differentiated by M-CSF showed predominantly antifibrotic activity because of pronounced MMPs production, while GM-CSF-induced cultures, on the contrary, were characterized by profibrotic properties due to the high level of TIMP-1 and type I collagen. M1, M2a, and M2c, induced by M-CSF, differed only in MMP-2 production, and M2a produced this metalloproteinase more than other subtypes. In the case of GM-CSF-differentiated cells, a higher level of production of TIMP-1 and, to a lesser extent, type I collagen was characteristic of M1, whereas M2c have minimal concentration of them among GM-CSF-induced macrophage subtypes. Concerning the level of cathepsin L production was relatively constant and did not depend on the generation conditions (differentiation and polarizing signals). Thus, the data obtained help to identify macrophage subtypes with anti- or profibrotic potential and may be useful for the development of cell therapy for diseases associated with fibrogenesis dysregulation.

Recently, there has been a steady increase in depressive disorders, which occupy an important place in the structure of the causes of disability. In the pathogenesis of depression, an important role is played by neuroinflammation, which is associated with impaired adult neurogenesis. Notably, neuroinflammation is partially reversible, and the leading role in the initiation and regulation of neuroregeneration is given to macrophages. Opposite states of macrophage activation are classically activated M1 and alternatively activated M2 macrophages, characterized, respectively, by pro- and anti-inflammatory activity. A balance shift towards M2 macrophages has been considered as a new therapeutic strategy of psycho-neurological disorders. One of the inducers of the M2 phenotype is the efferocytosis. We have previously developed an original protocol for the generation of human macrophages under conditions of deficiency of growth / serum factors, in which M2 phenotype is formed through efferocytosis. Macrophages (M2(LS), LS – Low Serum) obtained according to this protocol express M2-associated markers, and are characterized by high production of growth and pro- angiogenic factors (IGF-1, VEGF, BDNF, EGF, FGF-basic, etc.), which can suppress inflammation and stimulate neuroregeneration / neuroplasticity. In the model of stress-induced depression, the antidepressant effect of soluble factors of M2(LS) macrophages was shown, accompanied by a decrease in the level of pro- inflammatory cytokines in certain brain structures. However, the effect of M2(LS) factors on neurogenesis remained unexplored. In the present work, which is a continuation of the aforementioned study, we analyzed the effect of intranasal administration of M2(LS) soluble factors on neuronal density in different brain areas – the frontal cortex and hippocampus – of depression-like mice. The results obtained showed that neuronal density in the frontal cortex, CA1 and CA3 zones of the hippocampus, was significantly higher in mice with intranasal administration of M2(LS) conditioned medium than in depression-like mice, and reached the level of neuronal density in intact animals. These results may indicate the neuroregenerative activity of M2(LS) macrophages in the model of stress-induced depression, which is mediated through soluble factors and manifests itself in an increase in the density of neurons in the brain.

T cells expressing checkpoint receptors PD-1, TIM-3 etc., are potential targets for monoclonal antibody immunotherapy in multiple myeloma (MM). However, checkpoint expressing T cell compartment includes different subsets, and their dysregulation following anti-checkpoint therapy can lead to the development of adverse events.The aim of this study was to evaluate activation markers – homeostatic cytokine receptors and transcription factors expressed by PD-1and TIM-3-positive T cells.Material and Methods. Relative counts of circulating PD-1and/or TIM-3-positive and negative T cells expressing common ɣ-chain cytokine receptors CD25, CD122, CD127, phosphorylated STAT5, and transcription factor EOMES associated with T cell exhaustion were studied using flow cytometry in 17 healthy donors, 22 MM patients with remission and 7 MM patients with progressive disease.Results. T cells expressing PD-1 and/or TIM-3 inhibitory checkpoint receptors in MM patients consisted of CD25+EOMESactivated cells, CD4+CD25+CD127-FOXP3+ regulatory T cells (Treg), CD4+CD25-EOMES+ dysfunctional cells. CD25+ T cells from healthy donors and MM patients, regardless of the expression of the studied checkpoint receptors, were EOMES-negative. No such association was found for CD122 and CD127 cytokine receptors. EOMES is a marker of T cell exhaustion for CD4+ T cells, but not for CD8+ T cells, in which it is more associated with activation. The proportion of CD4+ Tregs among circulating PD-1+ and TIM-3+ T cells was relatively low. A higher content of cytokine receptors in the population of TIM-3+ T cells may indicate the predominant involvement of TIM-3 in the control of homeostatic proliferation of mature T cells under lymphopenic conditions, while the expression of PD-1 may be more associated with the regulation of activation through T cell receptor. PD-1+ and/or TIM-3+ levels of activated, dysfunctional, and regulatory T cells can potentially be used to predict the safety and efficacy of targeted immunotherapy.

Introduction. Myeloid-derived suppressor cells (MDSCs) play an important role in restriction of the immune response and are associated with a poor prognosis in cancer. Mobilization of hematopoietic stem cells (HSCs) before high-dose chemotherapy (HCT) with autologous HSC transplantation (auto-HSCT) is accompanied by a signifcant increase in MDSC counts in peripheral blood and apheresis product of multiple myeloma (MM) patients. However, quantitative changes of these cells at the post-transplant and their role at the immune recovery remain unexplored.The study was aimed to analyze the dynamics of circulating MDSC counts and the expression of suppressor molecule arginase-1 in patients with MM in the frst 12 months after HCT and auto-HSCT and evaluate association between MDSCs and transplantation outcomes.Material and Methods. The study included 44 MM patients who underwent HCT and auto-HSCT. The relative number of granulocytic MDSCs (G-MDSCs), monocytic MDSCs (M-MDSCs), and early-stage MDSCs (E-MDSCs), as well as the expression of arginase-1 in each of MDSC subsets was evaluated by fow cytometry in patient peripheral blood samples.Results. At the engraftment (day +12 – +16, leukocytes >1×109 /l), M-MDSC relative count was increased (pU=0.038), as well as the relative (pU=0.003) and absolute (pU˂0.0001) counts of G-MDSCs, decreasing after 6 months down to pre-transplant values (рU=0.007, рU=0.024 and рU=0.02, respectively) and remaining at the same level at the 12-month follow-up period. The absolute count of E-MDSCs by the time of the engraftment decreased transiently (pU=0.004 vs before HCT), gradually recovering by 12-month follow-up (pU=0.032 vs day +12 – +16). The remission within 12 months in the group with G-MDSCs˂0.17 % at the engraftment was observed in 67 ± 11 % of patients, with G-MDSCs >0.17 % – in 94 ± 6 % of patients (p=0.049). During the 12-month post-transplant, the number of M-MDSCs expressing arginase-1 has been increasing, with a tendency to lower values at the engraftment in patients with early MM relapse (pU=0.09).Conclusion. The association of early MM relapse after auto-HSCT with the lower count of G-MDSCs and the lower count of arginase-1+ M-MDSCs at the engraftment suggests that MDSCs is involved in the restriction of homeostatic proliferation as a factor for more effective immune recovery.

GM-CSF and M-CSF, the hematopoietic colony-stimulating factors, induce various phenotypic changes in macrophage lineage populations and promote cell differentiation, respectively, into M1- and M2-like macrophages. The pro- and anti-inflammatory properties of macrophages generated by these colony-stimulating factors are well described, but the contribution of differentiation and polarization signals to the fibromodulatory activity of macrophages remains unexplored. To clarify the differences in the fibrogenesis regulation mechanisms inherent in differently activated macrophages, we studied the effects of macrophage-conditioned media on proliferation and differentiation of dermal fibroblasts. In this study, the human macrophages generated from peripheral blood monocytes were investigated. They were induced for differentiation by M-CSF or GM-CSF, being further polarized in the M1 direction with lipopolysaccharide and, in the M2 direction, with IL-4 or dexamethasone. Proliferative response of the fibroblasts was determined radiometrically by [3H]-thymidine incorporation. Differentiation into myofibroblasts was determined with flow cytometry technique, as expression of a specific marker α-smooth muscle actin (α-SMA). The level of macrophage TGF-β1 production was assessed using an appropriate ELISA kit. The data obtained indicate that the macrophages differentiated under the influence of “homeostatic” M-CSF are characterized by a moderate stimulating effect upon fibroblast proliferation, and the effects of M2 (IL-4) and M2 (Dex) macrophages exceed that of M1 (LPS), but do not differ significantly from each other. The M-CSF-induced M1 (LPS) and M2 (IL-4) macrophages, but not M2 (Dex), enhance the fibroblast differentiation and show similar level of stimulation. In contrast to M-CSF, the macrophages induced by “pro-inflammatory” GM-CSF exhibit a pronounced stimulatory effect on fibroblast proliferation, and the effects of M2 macrophages exceed those of M1 cells, being most pronounced for M2 (Dex). At the same time, only GM-CSF-induced M2 (IL-4) macrophages enhance fibroblast differentiation. Dexamethasone-polarized macrophages do not significantly affect fibroblast differentiation regardless of the CSF used (M-CSF or GM-CSF). The content of TGF-β1 in the supernatants of differently activated macrophages does not correlate with the level of stimulating effect of macrophage-conditioned media upon fibroblast differentiation. In general, the data obtained suggest the involvement of differentiation and polarization signals into modulation of pro- and anti-fibrogenic properties of macrophages.

The aim of the present study was to assess safety and clinical efficacy of inhalation immunotherapy based on intranasal administration of bioactive factors produced by the M2 phenotype macrophages in children with language impairments, as well as to study the effect of inhalation immunotherapy on the cytokine profile in the patients' blood serum. The study was carried out according to the NCT04689282 protocol (www.ClinicalTrials.gov) and included 14 children (9 boys / 5 girls), aged 3 to 8 years, with language impairments associated with perinatal or postnatal CNS lesions of various origin. The children recruited into the study were assessed by a neurologist and speech therapist before the therapy, at the end of the course (1 month), and 6 months later. Serum samples for cytokine analysis were obtained before and 1 month after therapy. The course of intranasal inhalations by the conditioned M2 media (2 ml one time per day for 28-30 days) was safe and well tolerated. None of the 14 treated children had significant adverse reactions and severe undesirable events. Intranasal immunotherapy led to a decrease in the severity of language problems, which manifested by improved speech understanding by 45%; the sensorimotor level of speech, by 51%; word formation skills, by 72%, as well as a twofold increase in general and fine motor skills. In children with signs of autism spectrum disorders, along with a language improvement, a decrease in the severity of autistic symptoms was registered, as evidenced by statistically significant decrease in the CARS score from 42.5 to 38.5 after 1 month, and to 33 points after 6 months (p < 0.05). The clinical effect was revealed rather soon, i.e., within a month after the first procedure, being maintained or intensified during a follow-up for 6 months. At the same time, two-thirds of the children showed a clear clinical improvement, with insignificant effect in the rest of patients. Comparative analysis of the serum cytokine levels in these subgroups showed that children with a pronounced positive response to inhaled immunotherapy differed in the following parameters: (1) initially higher level of VEGF and IGF-1, and (2) decrease the level of TNFα in response to intranasal immunotherapy. In summary, we first tested a fundamentally new approach based on the use of soluble factors from M2-type macrophages and intranasal route of their administration in order to treat the children with severe language impairments, demonstrating safety and obtained preliminary data on effectiveness of such approach.

Expansion of myeloid-derived suppressor cells (MDSCs) due to impaired differentiation of myeloid progenitor cells under conditions of inflammation was described in a number of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes mellitus. Studying the role of MDSCs in ankylosing spondylitis is an important issue, given that increased concentration of proinflammatory mediators in this pathology can also cause myelopoiesis disorders. The aim of present work was to study the quantitative content of MDSC subpopulations in patients with different clinical phenotypes and activity of AS. 37 patients, including 10 patients without peripheral skeletal lesions (axial form) and 27 patients with simultaneous lesions of spine and peripheral joints (peripheral form) were recruited into the study. The control group consisted of 32 age/sex-related healthy donors. Evaluation of granulocytic (LinHLA-DRCD33+CD66b+; G-MDSC), monocytic (CD14+HLA-DRlow/-; M-MDSC) and early-stage MDSCs (LinHLA-DRCD33+CD66b- ; E-MDSC) was performed using corresponding antibodies (BD Biosciences, USA) in the population of peripheral blood mononuclear cells by flow cytometry. In general, the AS patients were characterized by an increased relative and absolute amount of M-MDSC (p = 0.00002 and p = 0.00003, respectively) and G-MDSC (p = 0.0002 and p = 0.0006, respectively). Patient gender, age, and HLA-B27 expression did not significantly affect the content of these cells in peripheral blood. An increase in the median values of M-MDSC was detected both in patients with axial (Ме 5.0 (3.2-6.3) versus 2.4 (1.7-3.5) %; p = 0.001) and peripheral form (Ме 5.0 (3.0-7.0) versus 2.4 (1.7-3.5) %; p = 0.0002) AS. At the same time, the G-MDSC expansion was observed only in patients with involvement of peripheral joints (Ме 0.16 (0.07-0.3) % versus 0.05 (0.04-0.09) %; p = 0.0001). The relative contents of E-MDSC, M-MDSC and G-MDSC in the axial form of AS was in direct correlation with the activity of the disease (R = 0.58, p = 0.02; R = 0.73, p = 0.08 and R = 0.65 p = 0.04, respectively). This relationship was not observed in peripheral form of AS. The data obtained suggest a potential involvement of MDSCs in pathogenesis and phenotypic heterogeneity of AS. Simultaneously, the revealed direct correlation between the MDSC contents and the disease activity suggests a decrease in suppressive activity and/or appearance of pro-inflammatory activity in MDSC, thus requiring further research in the field.

This article is the second communication in a series of articles devoted to the effects of a domestic preparation of macrophage-activating factor (GcMAF-RF) and assessment of its biological properties. The aim of this work was to study the effect of the GcMAF-RF upon M0 → M1 polarization of macrophages (Mph), and activation of the professional properties of ex vivo generated antigen-presenting dendritic cells (DC), as well as on ex vivo production of pro-inflammatory (TNFα, IL-1β, IL-6, IFNγ, IL-17, IL-18) and anti-inflammatory (TGF-β, IL-4, IL-10) cytokines, growth factors (IL-2, GM-CSF, G-CSF, VEGF) and chemokines (MCP, IL-8) by the whole blood cells from healthy donors. Mph and DC were generated from the monocytes (3 to 5×106 /ml) derived from adherent fraction of peripheral blood mononuclear cells (MNC) of healthy donors. Granulocyte/macrophage colony-stimulating factor (rhGM-CSF) was used to obtain Mph, whereas DC production was induced by GM-CSF and interferon-α. To provide M1 polarizing signals, bacterial lipopolysaccharide (LPS from E. coli 0114:B4) was used in controls. In experimental series, GcMAF-RF was added 48 h before the end of culture. The stimulating effect of the obtained Mph and DC upon cell proliferation was assessed in allogeneic mixed culture of leukocytes (alloMLC) using radiometric technique, by 3 H-thymidine incorporation. The influence index (IR) of Mph or DC upon allo-SCL was calculated as the ratio of the proliferative response of MNCs in the presence of Mph, or DC to the level of spontaneous MNC proliferation. To determine the cytokine production by human whole blood cells ex vivo, peripheral blood samples from 3 donors with two replicate GcMAF-RF preparations were used, at a total of 6 points. All variants of the study were carried out with mitogen-activated and non-activated blood cells. The cytokine content was determined by the ELISA assays. The effects of GcMAF-RF were quantified as a fold increase (FI), i.e., the ratio of cytokine production in the presence of GcMAF-RF to the level of their spontaneous production. It was shown that the GcMAF-RF preparation was as effective, as lipopolysaccharide (LPS), the standard Mph and DC activator which induces polarization of differentiated M0-macrophages into M1 cells and final maturation of DCs, manifesting by a significant increase in their allo-stimulatory activity in a mixed leukocyte culture (allo-MLC). Moreover, GcMAF-RF stimulates production of numerous cytokines and chemokines (TNFα, IL-1β, IL-6, IL-18, IL-4, IL-10, GM-CSF, G-CSF, VEGF, IL-8), by blood cells (granulocytes, lymphocytes, monocytes), thus indicating direct participation of the macrophage activator GcMAF-RF in various immune processes. The domestic GcMAF-RF drug induces polarization of macrophages M0 → M1, final maturation of DCs and allostimulating activity of Mf and DCs, and is also able to effectively stimulate circulating blood cells to synthesize cytokines/chemokines with pro-inflammatory and immunoregulatory activities.

Background. Recent studies demonstrated immunosuppressive properties of vascular endothelial growth factor (VEGF-A) and identified VEGF-A as a key mediator of tumor-induced immunosuppression. Placental growth factor (PlGF) is another member of VEGF family in which dramatic elevation is associated with effective immune adaptation in successful pregnancy, whereas low concentrations are related to pregnancy complications resulting from the activation of immune system. Previously, we have shown that activated T cells express VEGF receptor type 1 (VEGFR-1), and PlGF inhibits T cell proliferation in VEGFR-1–dependent manner.The aim of the present study was to further characterize PlGF effects on T cell responses in vitro.Materials and methods. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated with anti-CD3 monoclonal antibodies (a-CD3) in the absence or presence of PlGF and assessed for IL-10 production, programmed cell death and the expression of inhibitory receptors (PD-1, CTLA-4, Tim-3) in CD4+ and CD8+ T cell subsets.Results. The addition of PlGF to PBMC cultures activated with a-CD3 resulted in increased percentages of IL- 10-producing CD4+ and CD8+ T cells. Besides, PlGF promoted CD8+ T cells apoptosis while did not affect programmed cell death within CD4+ T cells. Notable, T cell activation with a-CD3 in the presence of PlGF was accompanied by the enhancement of PD-1-expressing cells in CD8+ T cell subset and Tim-3-expressing cells in both CD4+ and CD8+ T cells, and by the increased expression of PD-1 and Tim-3 on T cells.Conclusion. Our in vitro findings indicate that PlGF can inhibit T cell responses due to the increasing interleukin-10 (IL-10) production, promoting CD8+ T cell apoptosis and enhancing the expression of PD-1 and Tim-3 inhibitory receptors. Given the elevated levels of PlGF in successful pregnancy and its decrease in gestation complications, the obtained data also suggest that PlGF-mediated suppression may be implicated into the governing immune evasion in pregnancy.

Myeloid dendritic cells (DCs) play an important role in the immune response; therefore, the search for compounds that can effectively activate DCs is a needful goal. This study was aimed to investigate the effect of synthetic CpG oligodeoxynucleotides (CpG-ODN) on the maturation and allostimulatory activity of myeloid DCs in comparison with other PAMP and DAMP molecules. For the research, we synthesized known CpG-ODN class C (SD-101 and D-SL03) containing thiophosphate internucleotide groups, and their original phosphate-modified analogues (SD-101M and D-SL03M) with mesylphosphoramide internucleotide groups (M = μ-modification). The effects of CpG-ODN and other activators were evaluated on DCs generated from blood monocytes in the presence of GM-CSF and IFN-α (IFN-DC) or IL-4 (IL4-DC). Evaluation of the intracellular TLR-9 expression showed that both types of DCs (IFN-DC and IL4-DC) contained on average 52 and 80 % of TLR-9-positive cells, respectively. The CpG-ODNs studied enhanced the allostimulatory activity of IFN-DCs, and the effect of μ-modified CpG-ODNs was higher than that of CpG-ODNs with thiophosphate groups. The stimulating effect of CpG-ODN at a dose of 1.0 μg/ml was comparable (for D-SL03, D-SL03M, SD-101) with or exceeded (for SD-101M) the effect of LPS at a dose of 10 μg/ml. At the same time, IFN-DCs were characterized by greater sensitivity to the action of CpG-ODNs than IL4-DCs. The enhancement of DC allostimulatory activity in the presence of CpG-ODNs was associated with the induction of final DC maturation, which was confirmed by a significant decrease in the number of CD14+DC, an increase in mature CD83+DC and a trend towards an increase in CD86+DC. Interestingly, the characteristic ability of LPS to enhance the expression of the co-stimulatory molecule OX40L on DCs was revealed only for the μ-analogue SD-101M. In addition, CpG-ODNs (SD-101 and SD-101M) had a stimulatory effect on IFN-γ production comparable to the action of LPS. The data obtained indicate a stimulating effect of CpG-ODN on the maturation and allostimulatory activity of human myeloid DCs, which is more pronounced for μ-modified analogs.

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.

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+CD25hiT 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+CD25hiTreg, 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+CD25hiT 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+CD25hiT cell percentage and 4-fold higher CD4+IFNγ+/CD4+CD25hi ratio compared with opposing group. ROC analysis revealed a “good” quality of prognosis based on evaluation of the CD4+IFNγ+/CD4+CD25hi 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+CD25hiT 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.

Dendritic cells (DCs) play a key role in maintaining the peripheral tolerance of lymphocytes to autoantigens. Recovery of immunological tolerance in autoimmune diseases, particularly, in rheumatoid arthritis (RA) is considered a new therapeutic strategy. The aim of this work was to study the effect of dexamethasone-modified DCs generated from monocytes of RA patients in the presence of IFNα (DCsDex), upon autologous T lymphocytes in mixed leukocyte culture (auto-MLC), and to investigate possible mechanisms of the DCsdex tolerogenic effect upon autoreactive T cells. We have shown, that DCsDex from RA patients induce T cell hyporeactivity in auto-MLC. Hyporeactivity of T cells is associated with cell cycle blockage in CD4+T lymphocytes and decreased IFNγ, IL-17, IL-4 and IL-13 production, which indicates the induction of CD4+T cell anergy. In this case, inhibition of Th1/Th17 has been more pronounced than the suppression of Th2 cells producing IL-4 and IL-13. Along with T cell anergy, the decrease of proliferative response in auto-MLC is associated with increased CD3+T lymphocyte apoptosis. In addition, the DCsDex of RA patients suppresses the proliferation of autologous T cells stimulated by unmodified DCs. This effect is associated with enhancement of IL-10-producing CD4+T cells in the auto-MLC, thus being indicative for an ability of DCsDex to induce conversion of CD4+T lymphocytes into regulatory T cells (Tr1). The data obtained characterize a new type of tolerogenic DCs, generated from blood monocytes of RA patients in the presence of IFNα and modified by dexamethasone, thus revealing a mechanism for tolerogenic effect of DCsDex upon T cells that recognize self-antigens in auto-MLC.

The immunomodulatory activity of vascular endothelial growth factors (VEGFs) reveals a new role of neoangiogenesis in tumor development. Most of VEGF effects on T cells are mediated through the VEGF-R2 receptors. Placental growth factor (PlGF) belongs to the VEGFs family and is a selective ligand for VEGF-R1. In order to study the role of VEGF-R1-signaling in the regulation of T-cell functions, the effect of PlGF on the proliferation of donor T cell has been investigated. PlGF has been shown to inhibit the proliferation of T-lymphocytes in cultures of anti-CD3-stimulated mononuclear cells in a wide dose range, suppressing the proliferative response of both CD4 + and CD8 + T cells. The suppressive effect of PlGF was mediated through the direct interaction with VEGFR-1 on T-cells that was evidenced by the expression of VEGFR-1 by T-lymphocytes (especially after their activation) and by blocking the suppressive effect of PlGF with neutralizing anti-VEGFR-1 antibodies. Given the increased levels of PlGF in many tumors, this factor may play an important role in immunomodulation during tumor growth, mediating its effect through the VEGFR-1 signaling pathway.

Vitamin D3 Binding Protein (DBP) is a multifunctional glycoprotein whose main role is to transport vitamin D3 and its metabolites, but it also is the precursor of the macrophage activating factor (GcMAF). DBP is converted to GcMAF as a result of site-specific selective deglycosylation under the action of β-galactosidase and sialidase, localized on activated B and T cells, respectively. GcMAF exerts its biological activity primarily as the capability of activating macrophages by enhancing their phagocytic function and producing ROS. Activation results in elevated expression of the specific macrophageal surface receptors involved in the recognition of tumor-associated antigens, as well as in the implementation of direct anticancer activity by inducing the apoptosis or necrosis of tumor cells. Increased interest in GcMAF is associated with its potential to be used in the clinic as a new antitumor drug. Besides its anti-tumor activity, GcMAF exerts a potential against a number of viral and neurodegenerative diseases associated with increased activity of N-acetylgalactosaminidase (nagalase) in the blood serum of patients. Nagalase is an enzyme that completely (rather than selectively) deglycosylates DBP so it cannot be converted to GcMAF, leading to immunodeficiency. Circulating DBP is composed of unmodified and O-glycosylated molecules with the glycosylation degree being dependent on the allelic variants of the gene encoding DBP. The role of DBP in the resistance of organism against a number of diseases is supported by the increased risk of a variety of severe illnesses (amyotrophic lateral sclerosis, colorectal cancer etc.) in patients deficient for GcMAF due to homozygosity for defective DBP alleles. In this review, we also will examine in detail the current data i) on the structure and functions of DBP, as the main precursor of GcMAF, ii) on the main mechanisms of GcMAF anticancer effect, iii) on the tumor strategy for neutralizing GcMAF activity, iv) on the results of GcMAF clinical trials in various cancers; and will discuss the available controversies regarding the positioning of GcMAF as an effective antitumor drug.

The induction of a strong multi-epitope T-cell response against hepatitis C virus (HCV) plays an important role in eliminating the virus, whereas adoptive response deficiency contributes to chronic and rapid progression of HCV-infection. Since dendritic cells (DCs) are capable of priming naive T lymphocytes and induce an effective immune response, the use of DC-based vaccines to enhance the HCV-specific T cell response is considered as a new approach to treatment of chronic hepatitis C (CHC). The ability of DCs generated from monocytes in the presence of interferon-α and loaded with recombinant HCV proteins Core (1–120) and NS3 (1192–1457) to induce an antigen-specific cellular response in healthy donors and patients with CHC was investigated. The immune response was assessed by proliferative activity and Th1 (IFNγ)/Th2 (IL-4, IL-6) production in mononuclear cells (MNC) cultures, and activation of cytotoxic T-lymphocytes in the degranulation test. We demonstrated that the primary antigen-specific response in MNC cultures of seronegative donors was detected better by stimulation of DCs, loaded with both antigens (DCCore /NS3) than when loaded with a single protein. DCCore/NS3 induced the proliferative response and degranulation of CD8+ T cells in MNC cultures of all tested donors, and in 50% (5/10) cases — IFNγ production. Similarly to donor DCs, DCCore/NS3 of patients with CHC induced a proliferative response in most cases (86%) and IFNγ production in 57% cases. At the same time, the activation of cytotoxic T cells in patients was less frequent (patients vs donors 57 and 100%, respectively), which could be partly due to increased spontaneous degranulation of CD8+ T cells in some patients. The obtained data testify the possibility of using vaccines based on interferon-α-induced DCs for the prevention and treatment of chronic HCV infection.

The aim of the study was to analyze the relationship between monocyte subpopulations and phenotype/ functions of monocyte-derived dendritic cells (DCs), as well as DC sensitivity to the tolerogenic effect of dexamethasone.Materials and methods. The study included 15 healthy donors. DCs were generated by cultivating enriched fractions of CD14+ monocytes with or without CD16+cell depletion (CD16-Mo-DCs or CD16+Mo-DCs, respectively) in the presence of interferon alpha (IFNα) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte subpopulations were obtained by immunomagnetic negative selection.Results. CD16+Mo-DCs were characterized by higher percentage of mature (CD83+CD14-) and lower number of semi-mature (CD14+CD83+) cells, but were similar to CD16-Mo-DCs by HLA-DR and CD86 expression, involved in the presentation of antigens and activation of naive T-cells. and also to co-inhibitory/ tolerogenic molecules B7-H1 and TLR-2. CD16+Mo-DCs displayed higher allostimulatory activity, which was positively correlated with CD86 expression (rS = 0.69; p = 0.027) and negatively – with TLR-2 expression (rS = -0.72; p = 0.1). Allostimulatory activity of CD16-Mo-DCs was positively correlated with the number of mature CD14-CD83+DCs and semi-mature CD14+CD83+DCs. Addition of dexamethasone (10-6 M) into CD16-Mo-DCs and CD16+Mo-DCs cultures led to the delay of DC maturation, the decrease of CD86 and the increase of TLR-2 expression, as well as the increase of cells with co-inhibitory CD86- B7-H1+ phenotype that was positively correlated with the reduction of DC allostimulatory activity. The decrease of CD86+/TLR-2+ index in CD16+Mo-DC population was due to the reduction of CD86+DCs and in CD16-Mo-DC population – to the increase of TLR-2+cells. Dexamethasone possessed higher inhibitory effect on DC maturation in the CD16+Mo-DC cultures.Conclusion. CD14+ monocytes, both contained and depleted by CD16+ cells, can differentiate into DCs when cultured with IFNα. The presence of CD16+ cells in whole blood monocyte pool is associated with generation of DCs showed a more mature phenotype and higher allostimulatory activity. Both CD16- and CD16+ monocyte-derived DCs are sensitive to suppressive effect of dexamethasone. However, dexamethasone tolerogenic effect involves different mechanisms in CD16-Mo-DCs and CD16+Mo-DCs.

Primary cultures of human glioblastoma were obtained from the surgical material of patients K. (female, 61 years, Ds: relapse of glioblastoma) and Zh. (female, 60 years, Ds: relapse of glioblastoma). The effectiveness of a new therapeutic approach aimed at destroying the cancer cell community was evaluated on the primary cell lines of human glioblastoma culture by employing a new strategy of tumor-initiating stem cell synchronization and a domestic strategy of their eradication "3+1". The key elements of the strategy were the following indicator results: (1) evaluation of the presence of tumor-initiating stem cells in a population of cells from analyzed cultures by their ability to internalize double-stranded labeled DNA (TAMRA+ cells); (2) determination of the reference time points of the repair cycle of DNA interstrand cross-links induced by cross-linking cytostatic mitomycin C; (3) evaluation of cell cycle synchronization; (4) determination of the time (day after therapy initiation) when TAMRA+ cells were synchronously present in phase G1/S of the cell cycle, sensitive to the therapy; and (5) establishment of the TAMRA+ (tumor-initiating stem cells) eradication schedule. The cultures were treated with cross-linking cytostatic mitomycin C and a compositional DNA preparation. After the treatments, cell division slows down, and the cultures degrade. The K cell line completely degraded within 30 days of observation. The cell number of the Zh culture fell to nearly one-third of the starting value by day 15 of observation. On day 15, this indicator constituted 1/7.45 for mitomycin C and 1/10.28 for mitomycin C + DNA with reference to the control. The main target of the mitomycin C + DNA regimen was TAMRA+ tumor-initiating stem cells of the glioblastoma cell populations. The action of mitomycin C alone or in the combination with DNA demonstrated effective elimination of TAMRA+ tumor-initiating stem cells and the whole primary cultures of human glioblastomas.

The aim of present study was to evaluate safety and clinical efficacy of inhalatory immunotherapy based on intranasal delivery of bioactive factors produced by M2 macrophages applied for treatment of patients with organic brain syndrome (OBS).Materials and methods. The study under the NCT02957123 protocol (www.ClinicalTrails.gov) included thirty patients with OBS of various genesis (10 men and 20 women aged 18 to 81; Me, 62.5 years). Neurological assessment and the levels of 32 cytokines in the blood serum of patients were evaluated before and 2-3 days after completion of inhalation immunotherapy.Intranasal inhalations of cell-free culture medium of M2 macrophages (2 mL, once a day for 28-30 days) were safe and well tolerated. None of 30 treated patients had severe adverse events and serious treatmentrelated side reactions. One month after starting the inhalations, a positive dynamics in neurological status was noted in all the patients. A marked clinical response was documented in twenty out of thirty patients (67%), which manifested as improvement, according to all scales and questionnaires. The neurological improvement was not reversed over 6 months of follow-up period. In other ten patients (33%), a moderate clinical response was shown as improvement of individual scores. The positive changes were as follows: 1) a 43% decrease in anxiety and depression scores (according to HADS scale, pU = 0.0008); 2) an increase of total motor activity (stability and gait) by 25%, pU = 0.0001); 3) correction of cognitive functions (MoCa test, pU = 0.007); 4) reduced number and intensity of the disease symptoms by 52% (pU = 0.0001). This marked clinical response to immunotherapy is shown to be associated with correction/normalization of serum hepatocyte growth factor (HGF) level.Conclusion. Inhalation immunotherapy based on intranasal delivery of bioactive factors produced by M2 macrophages can improve neurological and functional recovery in patients with organic brain syndrome.

Dendritic cells (DCs) of patients with high-grade brain glioma exhibit impaired expression of membrane TNFα associated with low DC cytotoxicity against TNF-R1-expressing tumor cell line HEp-2. To assess the significance of these findings, we investigated a role of TNFα/TNF-R1-signaling pathway in DC cytotoxic activity against allogenic and autologous cell lines obtained from high-grade glioma tissues. DCs were generated by culturing of plastic-adherent peripheral blood mononuclear cells in presence of GM-CSF and IFNα for 4 d followed by LPS addition for 24 h (IFN-DCs). The tumor cell lines were obtained from tissues of 11 patients with glioblastoma multiforme. According our findings glioblastoma cells were sensitive to lysis mediated by donor IFN-DCs. The level of DC cytotoxic effect against cell lines obtained from different glioma patient tissues varied from 20 to 72.2%. DC lysis of 8 out of 11 glioblastoma cell lines exceeded 40%. Glioblastoma cell lines expressed both TNF-R1 and TNF-R2 receptors, but mostly – TNF-R1. However, rTNFα did not show cytotoxic activity towards glioblastoma cell lines. Blocking of TNFα/TNF-R1-signaling pathway by treating of donor IFN-DCs with soluble rhTNFR1 receptor led to partial decrease of DC cytotoxic activity against 5 out of 6 tested glioblastoma cell lines by 11-40% (median of suppression 24%). Glioblastoma patient IFN-DCs which were characterized by an impairment of TNFα/TNF-R1-signaling pathway lysed these glioblastoma cell lines, however median of DC cytotoxicity was 30% lower than that of donor values (31.5 vs 45.1%; р = 0.003). Cytotoxic activity of IFN-DCs of the glioblastoma patient against autologous tumor cells resistance to TNFα/TNF-R1-signaling pathway was comparable with level of cytotoxicity of donor IFN-DCs. Thus, glioblastoma cells are sensitive to cytotoxic activity of DCs mediated via TNFα/TNF-R1-signaling pathway, but the defect of this mechanism in IFN-DCs of glioblastoma patients determines significant decrease of DC cytotoxicity towards glioblastoma cells.

Objective. Mesenchymal stromal cells (MSCs) promote the differentiation of unprimed macrophages (Mр) or classically activated M1 cells towards alternatively activated, M2 macrophages. The aim of the work was to study the ability of MSC to induce M1→M2 switching by comparing the MSC effects on polarized M1 macrophages and monocytes stimulated by granulocyte-macrophage colony-stimulating factor to M1 differentiation.Material and мethods. MSC were co-cultured with monocytes for 7 days or M1 macrophages for 48 hours in Transwell system to prevent direct cell-to-cell contacts. To characterize generated Mр, classical M2 marker CD206, allostimulatory activity in a mixed lymphocyte culture (MLC), and the ability to secrete pro-/antiinflammatory mediators were analyzed.Results. Co-cultivation of MSCs and M1 macrophages led to the appearance of phenotypic (increased expression of CD206) and functional (decrease in allostimulatory activity) features of M2 phenotype. When MSCs were cultured with monocytes in the M1-inducing medium, generated Mр elicited a pronounced stimulating activity in MLC similar to that of M1 (stimulation index 3.45 and 3.4, p = 0.46) and significantly higher than allostimulatory activity of M2 cells (3.45 vs 2.2, p = 0.03). In addition, MSCs did not influence the expression of CD206, as well as the production of pro- (IL-1β, TNF-α, IL-6, IL-12) and anti-inflammatory (IL1-ra, IL-4, IL-10) cytokines, immunoregulatory cytokines (IFN-γ, IL-17) and chemokines (IP-10, MCP-1, MIP-1b, Rantes, Eotaxin).Conclusions: The ability of MSC to induce the M2 phenotype depends on the stage of differentiation of monocyte/macrophages. MSCs promote M1→M2 switching in cultures of polarized M1 macrophages. In contrast, when MSCs interacted with monocytes in M1-inducing medium, a population of M1-like macrophages is formed with high allostimulatory activity and typical for M1 spectrum of produced cytokines and chemokines.

Purpose of the study. Comparative study of the phenotypic and functional properties of dendritic cells (DC) in groups of patients with rheumatoid arthritis (RA) receiving disease-modifying drugs, or biological drugs and (or) pulse therapy with high doses of glucocorticoids.Materials and methods. The study included 39 patients with RA and 20 age-appropriate and semihealthy donors. Nineteen patients were treated with standard disease-modifying drugs (BMP) in the form of monotherapy or in combination (group PA1) at the time of the examination, 20 – biological preparations or pulse-therapy with glucocorticoids (group PA2). In the latter case, the examination was carried out for 2–7 days after the last injection of methylprednisolone.Results. In this study, the properties of DC generated from monocytes under the action of IFN-α (IFN-DK) for RA are described for the first time. It has been established that the general feature of DC in the PA1 and PA2 groups are signs of immaturity of the DC, manifested by increased CD14 expression and a decrease in the proportion of mature (CD14-CD83 +) DC. Despite the differences in DC in the PA1 and PA2 groups, both cell types retain in vitro sensitivity to dexamethasone, the treatment of which leads to a significant inhibition of TNF-α production and a decrease in allostimulant activity of the DC. Thus, IFN-DK in RA patients receiving medical therapy is characterized by the presence of tolerogenic properties, which are most pronounced when used in a program of treatment of biological agents or pulse therapy with corticosteroids.

Immune system plays a key role in pathogenesis of ischemic stroke. Ischemic brain injury causes destruction of cells and extracellular matrix proteins, and the release of „danger“ molecules that cause rapid activation of innate immunity, and induce adaptive autoimmune reactions. The cells of innate immunity are responsible for elimination of cellular and extracellular debris and tissue repair, being, however, involved in post-ischemic inflammation causing brain injury. An adaptive immune response which develops within a few days is directed against self-antigens, being not related to nervous tissue damage in acute phase of stroke. The cells of adaptive immunity are able to regulate microglial activity and promote repair processes in the central nervous system. However, long-term persistence of lymphocytes with reactivity for brain-derived antigens may be involved into the atrophy of cerebral cortex, and development of dementia in remote post-stroke period. Immune response following ischemic brain stroke is manifesting by local and systemic reactions. Nevertheless, in contrast to prolonged local intracerebral inflammation in infarction area, and peri-infarction region, a systemic inflammatory response is quickly replaced by development of immunosuppression, as evidenced by lymphopenia, lymphoid organ atrophy and defective functions of immune cells. Immunosuppression is largely due to activation of sympathoadrenal system, aiming for limitation of inflammatory and autoimmune reactions. However, excessive immunosuppression leads to development of infectious complications, which may often cause lethal outcomes, and also may have a negative impact on efficiency of repair processes. The present review provides experimental and clinical data describing the innate and adaptive immune reactions in response to cerebral ischemia. Moreover, this review discusses possible pathogenetic importance of immune cells and their positive or negative effects, thus allowing further development of new therapeutic approaches for treatment of ischemic stroke.

The paper presents results of clinical application of autologous stromal-vascular fraction (SVF) cells in patients with degenerative osteoarthritis (OA) of the knee, grade II and III (Kellgren–Lawrence scale).We recruited six patients with knee OA (3 men and 3 women; median age 64 years) with mean disease duration of 7 years. All the patients were administered a single intra-articular injection of autologous nucleated SVF cells at an average dose of 16.8±0.9 × 106 per joint (a total of 11 joints). The patients did not experience any serious side effects (allergic, toxic or inflammatory) related to the knee injection. Patient surveys at 1 month after SVF administration revealed a decrease in the severity of pain, as measured by a visual analog scale (VAS) and a specialized 100-point scale KOOS (subscale "pain") (p < 0.05 on both scales). Moreover, the patients reported improvement in the joint functions and quality of life related to affected joints on a KOOS scale (p < 0.05). These positive clinical changes persisted during 6 month follow up. Significant improvements were noted in ultrasound findings, with increased thickness of the cartilage layer at 3 months (in 73% of cases) and at 6 months (in 82%). Our pilot study demonstrated the safety and tolerability of intra-articular injection of autologous SVF cells in patients with moderate to severe OA. The results obtained also indicate a significant antiinflammatory effect of autologous adipose tissue SVF cells, which is manifested at the early stages of cell therapy. Our further investigations will be focused on exploring the SVF stimulatory effects on regeneration of damaged joints.

One of the most important properties of extracellular double-stranded DNA related to the treatment of various diseases is its ability to activate effector cells of the immune system (anti-tumor and vaccinal immunity) through dendritic cells (DCs). The stimulatory effect of DNA on DCs is mediated by the TLR9 signaling pathway and/or through a system of cytosolic sensors and is manifested by increased expression of MHC class II antigens and costimulatory molecules and by increased synthesis of immunoregulatory cytokines. In this work, the expression of cytokines, differentiation antigens and transcription factor genes has been investigated in DCs activated by double-stranded human DNA (i) without any additional factors, (ii) using a lipophilic agent, and (iii) by blocking TLR9 with chloroquine. Evaluation of the DNA effect was carried out after the 6- and 24-hour exposure. It was found that the preparation of double-stranded DNA transfected by Lipofectamine 2000 boosts DCs at the same level as Poly(dA : dT), a synthetic equivalent of double-stranded DNA. It was discovered that combined application of DNA and chloroquine enhances expression of the IFN-α, IFN-β, IFN-γ, IL­8, МСР1, VEGF, CD25, and CD83 genes by hour 24 of incubation. It was for the first time shown that genomic “self” double-stranded DNA as a mono agent activates mRNA synthesis of cytokines IFN-α, IFN-β, IFN-γ, IL­8, IL­10, and VEGF in DCs at 6 hours of induction.

Granule-mediated cytotoxicity of effector cells is a universal mechanism of tumor growth inhibition and induction of tumor cell death. The aim of present study was to evaluate expression of lytic molecules in DCs generated in presence of IFNα (IFN-DCs), and to analyze the role of granule-mediated mechanism for IFN-DC cytotoxic activity against tumor cell lines. IFN-DCs were generated by culturing of plastic-adherent peripheral blood mononuclear cells in presence of GM-CSF and IFNα for 4 d followed by LPS addition for 24 h. The tumor cell lines were obtained from malignant tissues from patients with glioblastoma multiforme. Maturation of IFN-DCs in presence of LPS was accompanied by accumulation of intracellular perforin and granzyme B molecules. Perforin expression showed a direct correlation with intracellular lysosome-associated membrane protein-1 (LAMP-1/CD107a) expression in LPS-stimulated IFN-DCs. However, CD107a expression did not increase under LPS stimulation. At the same time, LPS caused upregulated degranulation in IFN-DCs, as shown by an increase of surface CD107a expression on IFN-DCs. LPS activation of DCs generated from the same donors in the presence of GM-CSF and IL-4 (IL-4-DCs) did not influence perforin and granzyme B expression in IL-4-DCs which was significantly lower than in IFN-DCs. Intracellular pool of CD107a molecules was increased in response to LPS stimulation of IL-4-DCs, but surface CD107a expression did not change on IL-4-DCs. Studies of cytotoxic activity of LPS-stimulated IFN-DCs revealed that concanamicyn A (CMA), an inhibitor of vacuolar H+-ATPase and of perforin/granzyme B-mediated signaling pathway, caused reduced cytotoxicity of donor DCs towards glioblastoma cell lines. Involvement of perforin/granzyme B-signaling pathway into the DCs cytotoxicity was confirmed with glioblastoma cell lines, since blockage of this mechanism with vacuolar H+ ATPase blocker (CMA) caused inhibition of the IFN-DC cytotoxicity. Differently reduced DC cytotoxic activity by CMA may suggest that the glioblastoma cell lysis can be mediated via perforin/granzyme B-independent mechanisms.

Dendritic cells (DCs) play an important role in pathogenesis of rheumatoid arthritis (RA) and are considered a novel target for immune therapy. Under inflammatory conditions, local dendritic cells of non-lymphoid organs are thought to be differentiated from monocytes. Moreover, DCs differentiation and activation in RA may be largely controlled by interferon-alpha. The aim of the present study was to investigate phenotypic and functional properties of monocyte-derived DCs generated in the presence of interferonalpha (IFN-DCs) in RA patients, and to specify, whether IFN-DCs are sensitive to a tolerogenic effect of dexamethasone. Fourteen RA patients with moderate-to-high disease activity treated with disease-modifying drugs have been included into the study. Twenty sex- and age-related healthy donors were used as a control. IFN-DCs were generated from monocytes by culturing adherent fraction of mononuclear cells for 5 days with GM-CSF and IFNα in the absence or presence of dexamethasone (10-6 M). IFN-DCs from RA patients were characterized by increased numbers of CD14+CD83- and lower amounts of CD14- CD83+ cells, thus presuming a delayed maturation. Furthermore, IFN-DCs from patients were characterized by higher expression of B7-H1 and TLR2. The phenotypic changes did not significantly influence specific functional activities of DCs, in particular, the capacity of DCs to produce TNFα, IL-10, IL-6, to stimulate proliferation of allogeneic T-cells and to activate T-cells for Th1 and Th2 cytokine production. Generation of patients’ DCs in presence of dexamethasone caused a decrease in CD83 and CD86 expression, reduced TNFα production, and suppressed allostimulatory activity of the DCs. Moreover, dexamethasone inhibited the ability of DC to stimulate Th1 response, along with shifting the balance towards Th2-stimulating activity. The data obtained provide an evidence that IFN-DCs from RA patients remain sensitive to the tolerogenic effects of dexamethasone. Furthermore, the revealed variations in sensitivity of patient’s DCs to dexamethasone-mediated inhibitory effect may be of interest for prediction of therapeutic response to glucocorticoid therapy. Our results also provide an evidence for possible implementation of IFN-DCs as a new cell platform for obtaining tolerogenic DCs.

Evolution of existing paradigms on regenerative capacity of the central nervous system (CNS) and eventual role of immune cells in restoration of damaged nervous tissue offers new prospectives in treatment of neurological disorders based on immunotherapeutic approaches. Present review article addresses a role of macrophages in restoration of damaged CNS and provides data on functional heterogeneity of resident tissue macrophages (microglia), and monocyte-derived macrophages. We discuss possible ways of monocyte recruitment to CNS, relationships between microglia and recruited macrophages, as well as M1/M2 balance in neurological conditions. Moreover, the review proposes an experimental rationale for macrophage engagement into the CNS damage reconstruction, and concerns the mechanisms of macrophage regenerative activity. In summary, the data presented here allow us to suggest macrophages as a novel therapeutic target for suppression of neuroinflammation and enhancement of reparative processes. The first steps in the field are encouraging, with respect to clinical application of monocytes/macrophages or M1→M2 switching technologies for treatment of the neurological disorders, thus presuming a need for further research in this direction.

Previously, we reported on the development of a therapeutic regimen allowing eradication of primary murine Krebs-2 ascites transplants. This protocol involved multiple injections of dsDNA preparations administered during the NER and HR phases of repair of interstrand DNA cross-links induced by prior cyclophosphamide treatments. Mice treated under this protocol frequently developed secondary ascites, which indicated that some tumor-inducing cancer stem cells could survive the treatment and caused relapse. Further, we observed that animals receiving multiple dsDNA injections developed pronounced systemic inflammatory response. This prompted us to develop a more straightforward treatment regimen based on the synergistic activity of cyclophosphamide and dsDNA preparations, which would allow complete eradication of established primary Krebs-2 ascites and also be less toxic for the treated animals. This protocol relies on a precisely timed single injection of dsDNA during the NER/HR transition period of each repair cycle. Under this protocol, 8-day remission of Krebs-2 engrafted mice was achieved, which was similar to the results of the multiple-injection treatment schedule. We observed an increase in the average life span of Krebs-2- transplanted mice on a single-injection regimen, which was consistent with reduced toxicity of such treatment.

Cumulative evidence obtained in this series of studies has guided the logic behind the development of a novel composite dsDNA-based preparation whose therapeutic application according to the specific regimen completely cures the mice engrafted with otherwise lethal Krebs-2 ascites. The likely mechanism involves elimination of TAMRA+ tumor-inducing stem cells (TISCs) from Krebs-2 tumors. We performed quantitative analysis of TISC dynamics in Krebs-2 ascites following treatment with the cytostatic drug cyclophosphamide (CP) and untreated control cells. In intact ascites, TISC percentage oscillates around a certain value. Following CP treatment and massive apoptosis of committed cancer cell subpopulation, we observed relative increase in TISC percentage, which is consistent with reduced susceptibility of TISCs to CP. Nonetheless, this treatment apparently synchronizes TISCs in a cell cycle phase when they become sensitive to further drug treatments. We describe the regimen of synergistic DNA + CP activity against Krebs-2 ascites. This protocol results in a complete cure of 50 % of Krebs-2 engrafted mice and involves three metronomic injections of CP exactly at the timepoints when repair cycles are about to finish combined with dsDNA injections 18 hours following each CP injection. The “final shot” uses CP + DNA treatment, which targets the surviving yet highly synchronized and therefore treatmentsensitive cells. The first three CP/DNA injections appear to arrest Krebs-2 cells in late S-G2-M phase and result in their simultaneous progression into G1-S phase. The timing of the “final shot” is crucial for the successful treatment, which eradicates tumorigenic cell subpopulation from Krebs-2 ascites. Additionally, we quantified the changes in several biochemical, cellular and morphopathological parameters in mice throughout different treatment stages.

Chronic viral hepatitis C is the most common cause of liver damage and the global problem worldwide since is characterized by a high prevalence, high chronization rates, and significantly increases the risk of liver cirrhosis and hepatocellular carcinoma. Many studies have shown that antigen-specific СD4+ and CD8+T-cells play a key role in pathogenesis and outcome of the infection. While the strong sustained antigenspecific multi-epitopic T-cell response predicts successful viral elimination, a deficiency of adaptive immune response is associated with virus persistence. This review presents data about pathogenetic significance of T-cell response in viral elimination, viral persistence and hepatitis development. Possible mechanisms of T-cell response failure in chronic infection are discussed as well.

Type I Interferons are potent inducers of monocyte’s differentiation into dendritic cells (DCs). However, sensitivity of these DCs to tolerogenic effect of glucocorticoids has not been previously investigated. The aim of this study was to investigate the effect of dexamethasone upon maturation and functions of interferonalpha-induced DCs (IFN-DC) derived from healthy donors. DCs were generated from blood monocytes cultured for 5 days with GM-CSF and IFNα, in absence or with addition of dexamethasone (10-6 M), applied on the 3rd day. Addition of dexamethasone inhibited IFN-DC maturation, which manifested with increasing numbers of CD14+ cells and decreased percentage of CD83+ DCs. Dexamethasone did not significantly influence HLA-DR, CD86 and B7H1 expression. However, it caused a 2-fold increase of tolerogenic TLR-2 molecule expression. Along with suppression of IFN-DC maturation, dexamethasone inhibited production of proinflammatory/Th1 cytokines (TNFα, IL-1, IL-2, IFNγ, IL-12), and some chemokines (MIP-1α, RANTES). Dexamethasone-treated IFN-DCs exhibited a 2-fold lower allostimulatory activity in mixed lymphocyte culture (MLC). Worth of note, the capacity of IFN-DCs to stimulate T cell proliferative response in allo-MLC showed direct correlation with CD83 expression on DCs, and an inverse correlation with CD14 and TLR-2. Evaluation of Th1/Th2-polarizing activity of IFN-DCs showed that dexamethasone exerted a pronounced inhibitory effect upon ability of DCs to stimulate T cells for IFNγ production, along with lowgrade suppressive effect upon ability of DCs to induce IL-6 production, thus being indicative for a dominance of Th2-polarizing activity of IFN-DCs under the influence of dexamethasone. In general, the data obtained show that IFN-DCs are sensitive to tolerogenic action of dexamethasone, and, hence, the IFN-DCs may mediate the immunomodulatory effect of glucocorticosteroids and represent novel candidate cells for the development of therapeutic tolerogenic DC-based vaccines applicable for management of autoimmune disorders.

Chronic recurrent infections caused by herpes simplex virus (HSV) types 1 and 2 represent a serious medical and social challenge. Given an important role of immune system in surveillance of viral infections, an induced enhancement of antigen-specific immune response seems to be a promising approach to treatment of recurrent HSV infections, in particular, using dendritic cell (DCs) vaccines. The present paper contains results of an open pilot study evaluating efficacy and safety of dendritic-cell vaccines in the patients with recurrent HSV infections. Twenty-nine patients including 14 persons with labial herpes, and 15, with genital herpes have been enrolled in this study. DCs were generated in presence of GM-CSF and IFNα and then loaded with recombinant viral proteins (HSV1gD or HSV2gD). These cells were applied in 2 rounds of vaccination performed within 9 months. Immunotherapy with DCs did not induce sufficient side effects, and was accompanied by more than two-fold decrease in relapse rate and increased length of remissions during the 9-month period of treatment. Later on, we revealed a reduced frequency of relapses, and a 3-fold increase in remission duration over the subsequent 6-month follow up. The clinical effect during the treatment and at later terms (a half-year observation) was noted with both labial and genital herpes, as registered in the majority of patients, being associated with induction of antigen-specific proliferative response and normalization of reduced mitogenic responsiveness of mononuclear cells towards ConA. The patients’ survey at longer terms (more than 24 months) has shown that the beneficial effect of immunotherapy, in terms of reduced relapse rates, was maintained in 77.8% of the respondents by 48 months (a median follow-up term). At this time, the antigen specific proliferative response was maintained in 66.7% of patients. The data obtained suggest that the dendritic-cell vaccines may be a promising approach to the treatment of recurrent labial and genital herpes.

The objective of the present study was to assess the level of 26 cytokines secreted by peripheral blood cells of the patients with liver cirrhosis (LC; n = 20) during the cell therapy (CT). All the patients were administered with intravenously injected autologous bone marrow-derived mononuclear cells (MNCs) in a dose of 1.3±0.3 × 109 (Ме 1.0 × 109 ) followed by 14 days later intravenous injection of ex vivo generated mesenchymal stromal cells (MSCs) in a dose of 22.3±5.0 × 106 (Ме 16.0 × 106 ). The patients were examined before the CT, 2-3 days after the administration of MNCs and, then, 2-3 days after the introduction of MSCs. Cytokine-secretory function of peripheral blood cells was evaluated in a 24-hour whole blood cultures both in the absence of any stimulation and in response to lipopolysaccharide (LPS). The control group consisted of 10 healthy donors. The administration of patients’ bone marrow cells (both MNCs and MSC) was safe and well tolerated and caused no any adverse (toxic or allergic) events. Compared with donors, LC patients (especially, with class B+C by Child-Pugh) differed by an initially increased production of several cytokines and chemokines. Actually, there was a statistically significant increase of the spontaneous production of IL-9, MIP-1β, and IP-10, as well as a distinct trend to an increase in TNFα, IL-1ra, IL-4, IL-5, IL-6, IL-13, of MCP-1, MIP-1α, RANTES and Eotaxin. Moreover, the blood cells of LC patients were susceptible to the stimulatory effect of LPS, and the LPS-induced production of 11 out of 26 cytokines (IL-1ra, IL-6, IL-9, IL-15, IL-17, IL-7, IL-8, IP-10, MIP-1α, MIP-1β, Eotaxin) significantly exceeded the normative values. Transplantation of bone marrow MNCs had minimal impact on cytokine production. Meanwhile, the MSCs introduction resulted in a significant decrease in spontaneous and LPS-induced production of, respectively, 20 and 18 analytes including pro-/anti-inflammatory and immunoregulatory cytokines, chemokines and growth factors. The normalization of cytokine-secretory function following transplantation of MSCs revealed in the patients with LC indicates the weakening of an inflammatory activity of circulating blood cells and the decrease in their reactivity to endotoxin. MSC suppressive effect on cytokine production was dose-dependent, and most pronounced in patients with decompensated LC (class B+C by Child-Pugh) of viral etiology.

Existence of a small subset of cancer cells referred to as tumor initiating stem cells (TISCs) largely responsible for tumor progression and resistance to chemotherapeutic cytostatic drugs reperesent an important recent paradigm shift. The present work is the first report in the series of papers from our group where we describe the development of anticancer therapy based on the selective targeting of TISCs. Here were characterize a cytoreductive activity of cyclophosphamide (CP), double-stranded DNA (dsDNA) and combinations thereof against the TISC population present in mouse Krebs-2 ascites. We evaluated engraftment potential of Krebs-2 cancer cells treated in ascites-bearing mice in vivo, followed by re-engraftment to congenic recipient mice in a form of a solid graft. These data indicate that with our approach TISCs can be completely eliminated even from a well-established ascites. We demonstrate that dsDNA-internalizing and CD34-positive cells are more sensitive to the synergistic effects of CP and dsDNA. When Krebs-2 ascites are treated with human DNA 1-12 hours post CP injection, this results in either elimination of cells that internalize TAMRA-labeled DNA (TISCs) or alters their phenotype, which is accompanied with the loss of surface expression of CD34. Next, we show that the timepoint 18 hrs post CP treatment is critical to the ongoing repair process in that it divides the repair into two phases: nucleotide excision repair + dsDNA break repair and homologous recombination. Importantly, both of these phases can be conveniently used for targeting the tumorigenic potential of the graft. In the context of monotherapy, CP is most effective against ascites grafts when administered as serial injections. To achieve maximum efficiency, the timing of consecutive injections must match the time when cancer cells found at G2/M during the first injection enter G1/S and/or the time of active repair via homologous recombination.

In the present paper, we report on the series of experiments where multiple regimens of CP and dsDNA injections were tested for targeting the ascites form of murine Krebs-2 cancer in situ. We show that combining CP with cross-linked human and salmon dsDNA results in a synergistic toxicity for ascites-bearing mice, an observation supported by the histopathology analysis of organs and tissues of experimental animals. In contrast, using a composite mixture of native and cross-linked human and salmon DNA after CP injections leads to a significant increase in average lifespan of the treated mice. Further, we demonstrate that repeated rounds of CP+dsDNA injections result in dramatic anticancer effect. The timing of injections is chosen so that they target the cells that were insensitive to the previous treatments as they were in the G2/M phase. 3-4 rounds of injections are needed to eliminate the subpopulation of tumor-initiating cancer stem cells. Our experiments identified the regimen when complete resorption of the primary Krebs-2 ascites occurs in all of the treated animals, followed by a remarkable remission period lasting 7-9 days. Yet, this regimen does not prevent secondary site metastases (either solid or ascites form) from developing, which is likely caused by the migration of ascites cells into adjacent tissues or by incomplete eradication of cancer stem cells. To address these and other questions, we expanded the study and performed histopathology analysis, which indicated that secondary metastases is not the only cause of death. In fact, many animals displayed unfolding systemic inflammatory reaction which was culminated by multiple organ failure. Thus, we developed the concept for treating ascites form of Krebs-2 cancer, which allows elimination of the primary ascites.

Recent research revealed dendritic cells (DCs) to have direct antitumor cytotoxic activity and to inhibit the growth and proliferation of tumor cells in vitro. The aim of the present study was to investigate the association between the cytotoxic activity of dendritic cells generated in the presence of interferon alpha (IFN-DCs) and TNFα expression by IFN-DCs in patients with malignant lymphomas. It was shown that IFN-DCs of malignant lymphoma patients possessed low cytotoxic activity against tumor cell line HEp-2 associated with low expression of transmembrane TNFα (tmTNFα) and high level of soluble TNFα (sTNFα) secretion. Reduced DC cytotoxic activity and low tmTNFα expression on DC surface were observed mainly in Hodgkin’s lymphoma patients. In contrast, IFN-DCs of patients with non-Hodgkin lymphoma were endowed with the ability to lysis of HEp-2 cells and tmTNFα molecule expression was similar to that in IFN-DCs from healthy donors. It was determined that the increase of expression of tmTNFα molecule on lymphoma patient IFN-DCs induced by the addition of TNFα-converting enzyme inhibitor into IFN-DC cultures was associated with the enhancement of IFN-DC cytotoxic activity against HEp-2 cells.

Cytokines play an important role in the pathogenesis of liver cirrhosis (LC), determining the disease severity as well as hepatic and extrahepatic complications. The present study aims to characterize the cytokine profile of blood cells in 20 LC patients with regard to the stage (class A on Child-Pugh, n = 13, and the class B+C, n = 7) and the etiology of the disease (viral LC, n = 12 and the LC of non-viral etiology, n = 8) using cytokine 26-plex assay. The control group consisted of 10 healthy donors. Cytokine concentrations were tested both in spontaneous and bacterial endotoxin (LPS) stimulated 24 hour blood cultures. Four functional groups: pro-/anti-inflammatory cytokines (IL-1β, TNFα, IL-1ra, IL-10), immunoregulatory cytokines (IL-2, IFNγ, IL-12, IL-4, IL-5, IL-6, IL-9, IL-13, IL-15, IL-17), growth factors (G-CSF, IL-7, FGF-β, PDGF, VEGF) and chemokines (IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, Eotaxin) involved in the pathogenesis of inflammation, have been analyzed. The blood cells of LC patients were shown to be active producers of cytokines and were characterized by increased cytokine levels from all four functional groups. The enhancement of many cytokines was associated with the disease severity and viral etiology. The increased cytokine production in both intact and LPS-stimulated cultures indicated preserved reactivity of blood cells to endotoxin. Overall, the data obtained suggest the contribution of circulating blood cells in supporting of inflammatory response and the potential diagnostic/prognostic significance of blood cell-derived cytokines in patients with LC.

We have previously shown that acute traumatic brain injury (TBI) is accompanied by increased level of circulating bone marrow progenitors, and favorable outcome is associated with early mobilization of CD34+CD45+ hematopoietic progenitor cells (HP). The present study was aimed at investigating whether patients with early HP mobilization differed from those with mobilization failure by systemic inflammatory reaction and immune parameters. The TBI patients were characterized by increased levels of serum C-reactive protein (CRP), IL-1в, IL-6, IL-8, MCP-1, G-CSF and IL-1ra indicative for presence of systemic inflammatory response. Importantly, patients with lacking mobilization of early HPs were shown to have significantly higher serum levels of CRP, MCP-1, MIP-1в, and G-CSF and a lower level of VEGF. In addition, patients with lack of early HP mobilization differed by significantly lower absolute number of lymphocytes, CD3+ T cells, CD4+ T cells, CD16+ NK cells and proliferative response of mononuclear cells to stimulation with ConA as well as by 4-fold higher rate of infectious complications compared with the opposite group. These data suggest that correlation of early mobilization of CD34+CD45+ cells with a favorable outcome in TBI patients may be partially mediated by anti-inflammatory and immunomodulatory effects of circulating bone marrow progenitors.

Injections ofexogenous DNAin combination witha cytostatic agent cyclophosphamide (CP) cause illness anddeathofexperimental mice. This phenomenon is referredto this as delayeddeath. It has been foundthat upon intraperitoneal injections fragments ofexogenous DNAreachthe bone marrowniche andenter the nuclei ofbone marrowcells (BMCs). Also, fragments ofexogenous DNAare capturedfrom culture medium byBMCs generatedex vivo. Upon suchtreatment, the fragments are incorporatedinto cells in a non-degradedform. We estimate that under suchconditions upto 1800 kb ofexogenous DNAcan be present in BMCs as fragments. After ex vivo incubation withBMCs, fragments ofexogenous DNAinternalizedin cells ofbothintact andCP pre-treatedmice become circularized. It has been shown that fragments ofexogenous DNAcan form high-molecular weight structures in vivo. Moreover, fragments localizedin BMCnuclei are suggestedto integrate into chromosome(s) ofrecipient mouse genomes when treatedwithCP andexogenous DNA.

Mice were observed to get sick and die upon administration of exogenous DNA in a specific period of time following their pretreatment with the cytostatic cyclophosphamide (CP) (Dolgova et al., 2011). It was established that exogenous DNA reaches internal compartments of bone marrow cells (BMCs) where it is processed (Dolgova et al., 2012a). Thus, BMCs appear to be the primary targets for the synergic action of these preparations (Dolgova et al., 2012b).In the present study, we show that the copy number for mouse interspersed genomic repeats decreases in the genome of mouse mononuclear cells as a result of interstrand cross-link (ICL) repair after pre-treatment with cytostatic CP. This phenomenon occurs within the time span from 18 to 24 h following CP injection, which corresponds to the final step in the repair of the majority of double-strand breaks (DSBs), as predominant intermediates in ICL repair. Injections of exogenous DNA in CP-pretreated mice preserve the copy number of interspersed repeats at the original level. Our results suggest that the fragments of exogenous DNA participate in ICL-induced DSB repair, thereby compromising the repair process.

The synergic action of the cytostatic drug cyclophosphamide (CP) and fragmented exogenous DNA causes illness and death in mice (Dolgova et al., 2011–2013). The observed «delayed death» effect was most clearly pronounced when the DNA preparation was administered 18 to 30 hours after CP treatment. This time span is designated as «death window».It was found that injections of exogenous DNA result in sustained increase in bone marrow cell (BMC) apoptosis, which occurs throughout the time of DNA administration (18–30 hours). Exogenous DNA, both allogeneic and belonging to various taxa induces BMCs apoptosis. Plasmid DNA has the greatest effect on apoptosis induction.The analysis of reduction and restoration of BMC subpopulations as the mice progressed to death revealed a virtually complete loss of the 12–20-mkm fraction of the cell population (about 3–4 % vs. 35–40 % in the control), which corresponds to the maximum leukopenia on day 3 after CP treatment. However, the relative amount of CD34+ hematopoietic stem cells (HSCs) from day 15 and till the end of the observation constituted 1,2–1,4 %, which corresponds to the wild-type range. Comparison of BMC smears from the sternal bone marrow of the CP and CP+DNA groups of mice indicates that the BMC populations isolated from CP+DNA animals lack young committed lymphopoiesis progenitor cells. Moreover, the affected mice had immature blast cell types in their blood, which was never observed in healthy or CP-treated mice. Pathological and morphological analyses show that starting from posttreatment day 9, mice that received CP+DNA preparations displayed pronounced morphological changes in their lungs, liver, pancreas, central and peripheral immune system organs, and brain. Most of the pathological changes observed are consistent with severe inflammatory response. This suggestion is proven by structural equivalents of functional involution of lymphoid organs, such as thymus, spleen, and lymph nodes.We speculate that the death of treated animals resulted from multiple organ dysfunctions caused by accidental involution of lymphoid organs and the systemic inflammatory response syndrome, both associated with injections of fragmented exogenous DNA into experimental animals within the «death window», which corresponds to the final step in the repair of the majority of CP-induced double-strand breaks.

The aim of present work was to evaluate the effect of mesenchymal stromal cells (MSCs) from lymphoma patients on the in vitro and in vivo homeostatic proliferation of lymphocytes. Our data have demonstratedfor the first time, that MSCs from lymphoma patient are able to enhance the in vitro proliferation of donor and patient mononuclear cells (MNCs) in response to IL-2 or IL-7. This effect was observed within a wide rangeof MSC-to-MNC ratios (1:50-1:2). Lymphocyte reactivity to IL-2 or IL-7 was found to be an important factor determining the stimulatory effect of MSCs. Ex vivo studies in the patients undergoing autologous hematopoietic stem cell transplantation (AHSCT) have shown that the patients co-transplanted with MSCs exhibited higher proportions of proliferating CD8+T cells (especially, memory cells) at the day of engraftment, than before AHSCT, as compared to the patients subjected to standard AHSCT protocols. Moreover, there was no an increase in apoptosis of naive CD4+T cells that was typical for standard AHSCT. These data indicate that the stimulatory effect of MSCs upon T lymphocyte proliferation following chemotherapy-induced lymphopeniaconcerns, mainly, the memory CD8+T cell population, whereas more efficient recovery of naive CD4+T cells is due to anti-apoptotic effects of MSCs.

Abstract. Some functional properties of IFNα-induced dendritic cells (DCs) from the patients with pulmonary tuberculosis (PT) were under study. It was revealed that production of IFNγ and IFNα by DCs of the patients was decreased, but TNFα production was preserved at normal level. NO production by DCs from the patients was also reduced. In addition, it has been shown, that DCs of PT patients were characterized by low allostimulatory activity in mixed lymphocyte reaction (MLR), and this defect was more marked in the patients with PPD-anergy. As compared to healthy donors, DCs from the patients with PT (in particular, with PPD-anergy) exhibited a decreased capacity for induction of intracellular expression and production of IFNγ by T-cells. However, DCs from the patients with PPD-anergy were able to stimulate intracellular expression of IL-4 in CD3+T-cell subset. Hence, the results suggest that functional activity of DCs in the patients with PT is altered, and some of these changes are distinctly associated with decreased antigen-specific response. (Med. Immunol., 2008, vol. 10, N 2-3, pp 151-158).

In present work, a comparative analysis of cytostatic and cytotoxic activity of IL-4 and IFNα-induced dendritic cells (IL-4- and IFN-DCs) has been performed in healthy donors and patients with brain tumors. Cytostatic activity was evaluated, as a capacity of DCs to inhibit growth of NK-resistant HEp-2 and A-549 tumor cell lines, whereas cytotoxic activity was measured by assessment of HEp-2 lysis. As compared with IL4-DC, IFN-DCs from healthy donors displayed a more pronounced cytostatic effect and similar cytotoxic activity against HEp-2 cells. In the patients with brain tumors, IFN-DCs were characterized by decreased cytotoxic activity. Meanwhile, DCs from the patients with malignant gliomas virtually did not exhibit a cytotoxic potential, whereas IFN-DCs from the patients with benign brain tumors retained their cytotoxic properties. A comparative study has shown that, in contrast to healthy donors, IFN-DCs from the patients with either malignant or benign brain tumors did not inhibit HEp-2 proliferation. The loss of DC-mediated cytostatic activity was not associated with decrease of CD123+ DCs (known as cells with maximal tumor-inhibitory potential), and, most likely, it resulted from functional changes in IFN-DC populations.

Abstract. The study deals with evaluation of dehydroepiandrosterone sulfate (DHEAS) effects upon phenotype and functions of dendritic cells (DCs), generated from blood monocytes of pregnant women by means of GM-CSF and IFN-alpha. It was shown, that initial supplementation with DHEAS (10-6 M, day 1 of culture) did not influence the pattern of DC subsets. Meanwhile, addition of DHEAS at the stage of DC maturation (last day of culture) is accompanied by a significant increase in mature CD83+ cells and activated DCs (CD25+), like as their precursors (CD1a+). Furthermore, the DCs generated in presence of DHEAS were characterized by marked allostimulating activity and decreased ability to downregulate the numbers of CD56+CD16+ NK cells. Hence, DHEAS promotes generation, maturation, and allostimulating activity of DCs, along with decreased negative regulation towards CD56+CD16+NK cell amounts. DHEAS-mediated changes in DCs’ phenotype and functioning are discussed as a possible mechanism of disturbed immunological tolerance to fetal antigens in pregnant women with suprarenal hyperandrogenia.

Abstract. The aim of present study was to evaluate efficacy of generation and characterization of regulatory T cells (Treg). The Treg were derived from peripheral mononuclear cells of healthy donors, using activation with anti-CD3mAb and IL-2, either in absence (model 1), or in the presence of immunosuppressive agents (vitamin D3 and dexamethasone, model 2). It was shown, that the Treg, obtained in model 1, are characterized by decreased anti-CD3-induced proliferative response and lack of suppressive activity. Treg, obtained in presence of vitamin D3 and dexamethasone, proved to be profoundly anergic. Such a state may be partially abolished by exogenic IL-2. These cells are also characterized by suppressive activity mediated by both cell-to-cell contacts and soluble factors, such as IL-10. Hence, the regulatory T cells generated in model 2, can be referred as inducible Treg (Tr1). Nevertheless, increased ratio of cells expressing CD4+FOXP3+ among Treg obtained by model 2, may also suggest an ability to generate natural regulatory cells. (Med. Immunol., 2008, vol. 10, n 2-3, pp 173-180).