[Population rearrangement of B-lymphocytes expressing chemokine receptors in patients with chronic obstructive pulmonary disease]. / Populiatsionnaia perestroika B-limfotsitov, ékspressiruiushchikh khemokinovye retseptory, u patsientov s khronicheskoi obstruktivnoi bolezn'iu legkikh.

To date, there are no drugs that can prevent progressive destruction of lung tissue and small airway fibrosis in patients with chronic obstructive pulmonary disease (COPD). Therefore, molecular mechanisms of this disease are being studied. The aim of this study was to determine the chemokine receptor expression pattern of B-lymphocytes from peripheral blood and airways of patients with COPD. Peripheral blood was collected from 51 smokers with COPD, 21 healthy smokers, and 20 healthy non-smokers. Seven smokers with COPD and 7 healthy smokers were recruited to undergo bronchoscopy with bronchoalveolar lavage (BAL). The expression of chemokine receptors CCR5, CCR6, CCR7, CXCR3, CXCR4, and CXCR5 on the surface of blood and BAL B-lymphocytes was determined by flow cytometry. It was found that the percentage of blood B-lymphocytes expressing chemokine receptors CCR5 and CXCR3 was higher in smokers with COPD compared with healthy smokers and healthy non-smokers. The percentage of CD27⁺ B-cells expressing CCR5 and CXCR3 receptors exceeded the proportion of CD27⁻ B-lymphocytes expressing these receptors in peripheral blood of patients with COPD and healthy controls. In smoking patients with COPD, the percentage of BAL B-cells expressing receptors CCR5 and CXCR3 was also increased compared with healthy smokers. There were no differences in the percentage of B-lymphocytes expressing receptors CXCR4, CXCR5, CCR6, and CCR7 in both peripheral blood and BAL between smokers with COPD and healthy smokers. A greater percentage of CD27⁻ B-lymphocytes than CD27⁺ B-cells expressed receptors CXCR4, CXCR5, CCR6, and CCR7 in the peripheral blood of smokers with COPD and healthy controls. The results of this study indicate a modification in the chemokine receptor profile of B-lymphocytes in COPD.

Nortriptyline Modulates the Migration of Peripheral Blood Lymphocytes and Monocytes in Patients with Chronic Obstructive Pulmonary Disease.

In the present study, the effect of nortriptyline (1 and 10 µM), budesonide (10 nM) and their combination on the migration of peripheral blood lymphocytes and monocytes from patients with chronic obstructive pulmonary disease (COPD) towards chemokines CCL5 and CXCL10 was evaluated by flow cytometry. Nortriptyline (10 µM), both alone and in combination with budesonide, inhibited the migration of T helper cells, cytotoxic T lymphocytes, NK cells and B lymphocytes towards CCL5 and CXCL10, as well as enhanced monocyte migration towards these chemokines. The combination of nortriptyline (1 µM) and budesonide suppressed the chemotaxis of lymphocyte subpopulations towards CXCL10, but not towards CCL5, more effectively than budesonide alone. The combination of nortriptyline (10 µM) and budesonide inhibited the migration of lymphocyte subpopulations towards CCL5 and CXCL10 and activated monocyte chemotaxis towards both chemokines more effectively than budesonide alone. The results of this study demonstrate the ability of nortriptyline alone to modulate the migration of peripheral blood lymphocytes and monocytes from patients with COPD and to potentiate the effects of budesonide.

[The effect of glucocorticoids in combination with azithromycin or theophylline on cytokine production by NK and NKT-like blood cells of patients with chronic obstructive pulmonary disease]. / Vliianie gliukokortikosteroidov pri ikh sovmestnom primenenii s azitromitsinom ili teofillinom na produktsiiu tsitokinov NK i NKT-podobnymi kletkami krovi patsientov s khronicheskoi obstruktivnoi bolezn'iu legkikh.

Chronic obstructive pulmonary disease (COPD) is characterized by reduced sensitivity of cells to the anti-inflammatory effects of glucocorticoids (GCs). Azithromycin and a low dose theophylline have a significant impact on molecular mechanisms leading to corticosteroid resistance. The aim of this study was to evaluate the ability of azithromycin and theophylline to enhance the anti-inflammatory effects of GCs on the production of cytokines by NK and NKT-like blood cells of COPD patients. Whole blood cells from COPD patients (n=21) were incubated in the presence of budesonide (10 nM), azithromycin (10 µg/mL), theophylline (1 µM), or their combinations and stimulated with phorbol myristate acetate (50 ng/mL). Intracellular production of proinflammatory cytokines in NK (CD3-CD56+) and NKT-like (CD3+CD56+) blood cells was analyzed by flow cytometry. Budesonide reduced synthesis of interleukin 4 (IL-4), CXCL8, tumor necrosis factor α (TNFα) by NK and NKT-like cells, as well as production of interferon γ (IFNγ) by NK cells. Azithromycin suppressed production of IL-4 and CXCL8 by NK and NKT-like cells, and theophylline inhibited IL-4 synthesis by these lymphocytes. The combination of azithromycin and budesonide had a more pronounced inhibitory effect on the production of IL-4 and CXCL8 by NK and NKT-like cells than the effect of these drugs alone. The combination of theophylline and budesonide suppressed synthesis of IL-4 and CXCL8 by NK and NKT-like cells, as well as the production of TNFα and IFNγ by NK cells stronger than budesonide alone. The obtained results demonstrate the benefits for the combined use of GCs with theophylline at a low dose or azithromycin to suppress the inflammatory process in patients with COPD.

Nortriptyline enhances corticosteroid sensitivity of blood T cells from patients with chronic obstructive pulmonary disease.

Steroid unresponsiveness is a significant problem in the management of chronic obstructive pulmonary disease (COPD). The tricyclic antidepressant nortriptyline has been reported to reverse corticosteroid resistance induced by oxidative stress. This study examined the potential synergistic anti-inflammatory effects of nortriptyline and corticosteroids in T lymphocytes from patients with COPD and the molecular mechanisms underlying their action. Peripheral blood mononuclear cells (PBMCs) or whole blood cells from COPD patients were incubated with budesonide, nortriptyline, or their combinations and stimulated with phytohaemagglutinin (PHA) or phorbol myristate acetate (PMA) plus ionomycin. The release of interleukin 4 (IL-4), IL-5, IL-8 and other mediators from PBMCs was measured by ELISA. Intracellular pro-inflammatory cytokines, glucocorticoid receptor (GR) and its isoform GRß, histone deacetylase 2 (HDAC2), phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) and p65 nuclear factor-κ (p65 NF-κB) were determined in CD4+ and CD8+ T cells using flow cytometry. Nortriptyline 10 µM decreased PHA-induced release of cytokines: IL-4, IL-5, IL-13, IL-17A, IL-33, macrophage migration inhibitory factor and thymic stromal lymphopoietin (TSLP). This drug alone also reduced the percentage of IL-4, IL-8, interferon gamma (IFNγ) positive CD4+ T cells and IL-4, IL-8 expressing CD8+ T cells stimulated with PMA/ionomycin, whereas nortriptyline 1 µM had less pronounced effect. The combination of budesonide 10 nM with nortriptyline 10 µM was more potent at suppressing IL-4, IL-5, IL-8, IL-13, IL-17A, TSLP secretion from PBMCs, as well as IL-4, IL-8, IFNγ, GRß expression by CD4+ and CD8+ T cells when compared with single budesonide treatment. The association of budesonide 10 nM and Nt (1 µM to 10 µM) effectively decreased p38 MAPK and p65 NF-κB phosphorylation and increased HDAC2 expression in both CD4+ and CD8+ T cells. In conclusion, nortriptyline alone demonstrates anti-inflammatory effects on blood T lymphocytes from COPD patients. Nortriptyline may also potentiate the effects of glucocorticoids and overcome corticosteroid insensitivity.

[Evaluation of Photo- and Cytotoxicity of Chlorin e6 Ester Derivatives and Their Liposomal Forms].

Photophysical characteristics and photosensitizing activity of the chlorin e6 dimethyl and trimethyl ester derivatives in various solution and their liposomal forms were studied. It was shown that in lipid vesicles chlorin e6 ester derivatives are predominantly in the monomeric state and possess optimal photophysical properties and high photochemical activity. The rate of redistribution of the chlorin e6 dimethyl ester from lipid vesicle to cells was higher as compared with that one of the chlorin e6 trimethyl ester. The increase of the serum concentration in the incubation medium has a different effect on processes of accumulation of the liposomal forms of the chlorin e6 dimethyl and trimethyl ester derivatives by the cells. Cell culture studies showed that application of liposomal forms of the chlorin e6 dimethyl and trimethyl ester derivatives significantly decreases their cytotoxicity but keeps high cytotoxic effect of photodynamic activity of the chlorin e6 ester derivatives.

Drug resistance associated properties of blasts subpopulations with different CD34 expression in childhood acute lymphoblastic leukemia (ALL).

AIM: The objective of this study was to investigate expression of drug resistance associated genes in CD34+ and CD34- leukemic subpopulations in childhood acute lymphoblastic leukemia (ALL). METHODS: ALL samples with heterogeneous CD34 expression were separated into CD34-positive and CD34-negative subpopulations and mRNA levels of MDR1, LRP, BCRP and BCL-2 genes were compared. RESULTS: BCL-2 gene expression levels did not differ significantly between CD34+ vs CD34- subpopulations in most analyzed ALL cases. Oppositely, MDR1 gene had >two-fold differences in expression levels between subpopulations in the majority of ALL cases. In T-lineage ALL CD34- fractions had increased level of BCRP and LRP genes in comparison with CD34+ ones whereas in most of B-lineage ALL expression of these genes did not differ. CONCLUSION: It was not found the unique pattern of resistance related genes expression in CD34+ vs CD34- subpopulations. However, in majority of studied pediatric ALL cases with CD34 heterogeneous expression one of subpopulations (positive or negative) could have an advantage for survival through elevated expression of drug resistance related genes.

Expression of MDR1, LRP, BCRP and Bcl-2 genes at diagnosis of childhood all: comparison with MRD status after induction therapy.

AIM: to investigate properties of leukemic cells by sorting out children diagnosed with ALL with different response to chemotherapy based on MRD level. METHODS: We used a minimal residual disease (MRD) data on day 36 obtained with 3-colour flow cytometry as a reference. In view of MRD results, we used real-time PCR to assess expression levels of multidrug resistance associated genes MDR1, LRP and BCRP, antiapoptotic gene Bcl-2 in initial samples from children diagnosed with ALL. P-gp expression and function in initial samples were analyzed by flow cytometry. RESULTS: Briefly, medians of relative expression levels of MDR1 gene were roughly comparable and in MRD(+) group came to 22.8 (0.02-26.6; n = 9) vs 24.8 (3.9-41.4; n = 10) in MRD(-) group. Bcl-2 gene showed tendency to higher expression levels in MRD(+) group with median at 5992.9 (521.0-10362.0; n = 9) compared to 3183.6 (1947.9-6581.0; n = 10) in MRD(-) group. LRP gene relative expression levels were similar in both groups and came to 1934.9 (1500.7-3490.4; n = 9) and 1408.5 (665.5-2917.1; n = 10) in MRD + and MRD(-) groups, respectively. The median of BCRP expression levels in MRD + group was considerably lower than that in MRD - group, namely 76000.0 (48196.2-169230.8; n = 9) and 227967.2 (16683.7-422222.2; n = 10), respectively, but statistical analysis showed no significant difference for this parameter. CONCLUSION: We investigated expression of multidrug resistance genes MDR1, LRP and BCRP and antiapoptotic gene Bcl-2 in leukemic cells at diagnosis, and MRD level at the end of induction therapy, and could not find obvious relations between these parameters.

Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro.

AIM: To test the ability of cultured mesenchymal stem cells (MSCs) from bone marrow (BM) of children with oncohematological diseases after chemotherapy and radiation to support proliferation and self renewal of hematopoietic cells in vitro. METHODS: BM samples of 8 patients and 9 healthy children-donors used for MSCs preparation applying technique of expansion in vitro. CD34+ cells were isolated from BM of donors. The ability of MSCs to maintain hematopoietic stem cells (HSCs) proliferation was tested in semisolid methylcellulose medium and in liquid long-term-culture (LTC) medium. RESULTS: The presence of MSCs derived from BM of patients in methylcellulose medium induced 2-fold increase of the number of committed myeloid progenitors without cytokines, 7-fold increase together with growth factors and 14-fold increase of the amount of earlier pluripotent hematopoietic precursor cells (CFU-GEMM) compared to expansion of HSCs without MSCs and cytokines. The presence of MSCs layer of patients in liquid LTC medium significantly promoted the hematopoietic cells proliferation rate, measured on 7th, 14th and 21st day. The total number of cells was multiplied 161.2-fold on 21st day as compared to 116.4-fold without MSCs layer. In the presence of MSCs layer, we detected the increase of proportion of bipotent CFU-GM precursors from 4% to 11% and pluripotent CFU-GEMM precursors from 0.1% to 0.6% in population of HSCs. In both types of experiments the capacity of patients' MSCs to support HSCs proliferation and self renewal was the same as for healthy donors' MSCs. CONCLUSION: In this study, MSCs were isolated from BM of children with malignancies after high-dose chemotherapy or radiation. The ability of these MSCs to maintain hematopoiesis in vitro was tested. It was shown that co-transplantation of autologous MSCs is a good way to improve hematopoietic stem cells engraftment and reduce a period of granulocytopenia after autologous HSCs transplantation in case of insufficient CD34+ cell number in autologous transplant.

Study of expression and functional activity of P-GP membrane glycoprotein in children with acute leukemia.

We studied expression and functional activity of tumor cell P-gp in children with various leukemia variants and analyzed its prognostic role in acute lymphoblastic leukemia. Functional activity of P-gp increased in acute myeloid leukemia, relapses of acute lymphoblastic leukemia (in comparison with primary disease), and in a group of patients in whom no remission was attained. The survival of patients with acute lymphoblastic leukemia was lower in cases with increased expression and function of P-gp.

[Drug sensitivity of tumor cells in varieties of acute childhood leukemia].

Drug sensitivity of tumor cells was studied in 154 children diagnosed with acute leukemia. Cellular sensitivity in acute lymphoblastic leukemia (ALL) was higher than in acute myeloid one (AML), while T-line ALL cells were more sensitive to dexamethasone than those of B-line. It was suggested that lower drug sensitivity of ALL cells at the earlier stages of cell differentiation was due to their reduced susceptibility to apoptosis. Among AML cells, M3 variant was less sensitive to cytozar while M4 - to vepeside. Reduced sensitivity to vincristine was found in series M3 > M1 - M2 > M4 while that to L-asparaginase was in M1- M2 > M4 > M3.

[Salvage thermochemotherapy in advanced refractory malignant tumors in children]. / Termokhimioterapiia pri refrakternykh i daleko zashedshikh zlokachestvennykh opukholiakh u detei.

The data are presented on polychemotherapy given to 17 children with advanced refractory malignant tumors using whole body hyperthermia and hyperglycemia. All patients suffered tumor progression throughout treatment and afterwards. Adjuvant Roncoleukin (interleukin-2) was administered in 5. Such salvage therapy was followed by overall tumor regression in 29.3%. Overall 4-year survival in such cases was 19%. Immunological monitoring of adjuvant whole body hyperthermia and interleukin-2 was carried out.

Different Mechanisms of Resistance to Lymphokine-activated Killer (LAK) Cells in Leukemic Cell Sublines Selected by Etoposide or by LAK Cells.

Two models of in vitro induction of human IM-9 lymphoblastoid cell line resistance to LAK cell-mediated killing were compared. IM-9 cell line variants were selected in vitro by prolonged exposure to LAK cells or to etoposide. Sensitivity to killing by LAK cells or by etoposide, conjugation with LAK cells and surface marker patterns were compared. Both LAK-cell-selected cell subline (IM-9/SL-15) and etoposide-selected cell subline (IM-9/ER) have acquired resistance to LAK cell-mediated death. IM-9/ER cells, but not IM-9/SL-15 cells, were 3.2-fold less sensitive to etoposide as compared to parental IM-9 cells. IM-9/SL-15 cells revealed decreased conjugation with LAK cells and augmented CDlla/CD18 surface molecule expression as compared to IM-9 line. In contrast, IM-9/ER cells displayed a level of conjugation with LAK cells equal to IM-9 cells, but loss of CD11a/C18 expression. Our results suggest different mechanisms of acquired resistance to LAK cells are operative in etoposide- or LAK-selected sublines, involving deterioration of LFA-1 molecule expression and altered conjugation properties, respectively.