Igor Kudryavtsev

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca(2+), and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

Dual proapoptotic and pronecrotic effect of hydrogen peroxide on human umbilical vein endothelial cells

Human umbilical vein endothelial cells were exposed in culture to hydrogen peroxide (H2O2), keeping them close to physiological conditions (high cell density, high serum content, H2O2 concentration not over 500 µM). Cell viability was assessed by flow cytometry using simultaneous staining with the fluorescent dye PO-PRO-1 to detect early apoptotic cells and DRAQ7 to detect late apoptotic and necrotic cells. The data obtained suggest that the primary mechanism of the cytotoxic response to H2O2 is apoptosis. The critical concentration of H2O2 causing death in a dense monolayer is 250 µM. Lower H2O2 concentrations (up to 200 µM) cause death of individual cells. The population of endothelial cell retains viability and response to calcium activating agonists does not change compared to control cells.

Molecular targets for the therapy of cancer associated with metabolic syndrome (transcription and growth factors)

Metabolic syndrome (MS) is one of the leading risk factors for the development of cardiovascular diseases, type II diabetes mellitus and reproductive system diseases. Currently, not only cardiovascular disease and reproductive history risks related with MS are frequently discussed, but it has been also shown that MS is associated with increased risk of some common cancers (endometrial cancer, postmenopausal breast cancer, colorectal cancer, biliary tract cancers and liver cancer for men). Further studies are required to understand the mechanisms of the involvement of MS components in the pathogenesis of malignant neoplasms. Changes in the expression of transcription and growth factors in the peripheral tissues as well as in cancer tissues of patients with MS were revealed. Transcription factors (AMP‐activated protein kinase‐1, STAT3, sterol regulatory element‐binding protein‐1 and peroxisome proliferator‐activated receptor‐γ), leptin and adiponectin receptors seem to be the most promising molecular targets for the therapy of cancers associated with MS.

Alkaloids of fascaplysin are effective conventional chemotherapeutic drugs, inhibiting the proliferation of C6 glioma cells and causing their death in vitro

Glioblastoma multiforme is an invasive malignant glial brain tumor with a poor prognosis for patients. The primary reasons that lead to the development of treatment resistance are associated with tumor cells infiltrating the brain parenchyma and the specific properties of tumor stem cells. A crucial research area in medical science is the search for effective agents that are able to act on these targets. Fascaplysin alkaloids possess potent antitumor activity. Modern methods for the targeted delivery of drugs reveal extensive possibilities in terms of the clinical use of these compounds. The aim of the present study was to establish effective concentrations of fascaplysin that inhibit the growth and kill the cells of glial tumors, as well as to perform a comparative analysis of fascaplysins effectiveness in relation to other chemotherapy drugs. C6 glioma cells were utilized as an optimal model of glioblastoma. It was established that fascaplysin at 0.5 µM has a strong cytotoxic effect, which is subsequently replaced by tumor cell death via apoptosis as the length of drug exposure time is increased. Fascaplysin kills glioma cells at a dose higher than 0.5 µM. The efficiency of fascaplysin was observed to significantly exceed that of temozolomide. Therefore, a significant feature of fascaplysin is its ability to inhibit the growth of and kill multipotent tumor cells.

Chapter 24 – Flow Cytometry and Light Scattering Technique in Evaluation of Nutraceuticals

Abstract Toxic and mechanistic properties of nutraceuticals are not as extensively studied as those of pharmaceuticals. Flow cytometry is among the most popular techniques to investigate the modes and mechanisms of cytotoxic action of chemical compounds, although flow cytometry methods are not suitable for online registration of fast changes of cell volumes. The latter is especially important for some types of cells, such as human erythrocytes with no nuclei and mitochondria. This chapter describes a new device (LaSca, Biomedsystems Ltd., Russia) for the registration of kinetic changes in cell volume, morphology, and aggregation. In addition, flow cytometry methods of cell viability assessment are overviewed, with attention given to mechanisms of cell death and survival, and some cellular studies of nutraceuticals using light scattering and flow cytometry techniques are summarized.

Cytotoxic Power of Hydrogen Peroxide Effect on Endothelial Cells in vitro

The currently existing terms “apoptotic index” and “apoptotic potential” are insufficient for the adequate qualitative evaluation and analysis of the proapoptotic activity of chemical agents within the wide range of therapeutic and toxic doses. Herein we introduce a new concept of cytotoxic power as an alternative to these terms related to apoptosis and other types of cell death on the basis of experimental data of hydrogen peroxide cytotoxic activity towards the human umbilical vein endothelial cells (HUVEC). We also propose an algorithm for computing cytotoxic power. Correlations between the percentage of early apoptotic and late apoptotic/necrotic cells and the expression or concentration of endothelial markers such as CD143, CD309, von Willebrand factor, phosphatase PTEN, have been revealed.

Apoptosis level in developing T cells in the thymus

163 The thymus as the central organ of the immune system is responsible for the maturation of T cells. It is assumed that the formation of the TTcell receptor (TCR) is accompanied by the destruction of over 95% of CD4 + CD8 + cells. This hypothesis is based on the results of studies obtained in 1960–1970 in vitro, which were extrapolated to the processes in vivo withh out experimental confirmation [1, 2]. The mathematt ical models of the processes developed to date, which are based on the existing hypothesis, predict the death of 75% of thymocytes in the course of selection [3, 4]. Solving the problem on the level of apoptosis in maturing T cells is essential for the validation of the model of their intrathymic maturation, including poss itive and negative selection as a prerequisite. The morphological analysis of intact mammalian thymus does not reveal the whole picture of masssscale apoptosis of maturing cells [5]. The use of flow cytomm etry also showed that the level of apoptosis in rat thyy mus reaches its maximum on prenatal day 18 (but accounts for only 25% of thymocytes), after which it drops to 5% and remains at this level in the postnatal period [6]. Due to the heterogeneity of published data and a great theoretical importance, the purpose of our work was to estimate the level of apoptosis in intact thymus by comparing the results of immunohistochemical analysis and the data obtained by flow cytometry. The study was performed with 6–100weekkold outt bred male mice and male CBA/JRap mice weighing 18–20 g. The animals were housed under standard vivarium conditions and received water and feed ad libitum. Work with animals were performed in accorr dance with the rules of the Commission on Bioethics of the Research Institute of Experimental Medicine. The level of apoptosis was assessed using panspee cific caspase inhibitors FAM FLICA Poly Caspase Assay Kit conjugated with fluorochromes [7] (Immuu nochemistry, United States); the results were expressed as percentage of FLICA + cells. The level of proliferating cells in the thymus was determined using the antibodies to the nuclear protein PCNA (BioLegg end, United States); the results were expressed as perr centage of PCNA + cells. The main populations of thyy mocytes were identified using the monoclonal antii bodies against mouse CD4, CD8, and CD45, which were conjugated with PE/Cy5, PE/Cy7, and APC/Cy7, respectively (BioLegend). All experiments were performed …

Purinergic Profiling of Regulatory T-cells in Patients with Episodic Migraine

Objectives: Immune responses in migraine are poorly characterized, yet implicated in the disease pathogenesis. This study was carried out to characterize purinergic profiles of T-cells in patients with episodic migraine without aura (MWoA) to provide mechanistic evidence for ATP and adenosine involvement in modulation of immune regulation in migraine. Methods: Peripheral blood samples were obtained from patients with migraine (n = 16) and age-matched control subjects (n = 21). Subsets of T-cells were identified by flow cytometry based on specific membrane markers. Results: Migraine patients showed reduced total T-cell counts in the peripheral blood. Whereas the total number of CD3+CD4+, CD3+CD8+, or regulatory T lymphocytes (Treg) was not changed, the proportion of Treg CD45R0+CD62L– and CD45R0–CD62L– cells was increased. Interestingly, in migraine, less Treg cells expressed CD39 and CD73 suggesting disrupted ATP breakdown to adenosine. The negative correlations were observed between the duration of migraine and the relative number of CD73+CD39– Tregs and total number of CD73-positive CD45R0+CD62L+ Tregs. Conclusion: Obtained data indicate that T-cell populations are altered in episodic migraine and suggest the involvement of Tregs in the pathophysiology of this disorder. Reduced expression of CD39 and CD73 suggests promotion of ATP-dependent pro-inflammatory and reduction of adenosine-mediated anti-inflammatory mechanisms in migraine.

NEUTROPHIL ACTIVATION IN RESPONSE TO MONOMERIC MYELOPEROXIDASE

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also regulate cellular functions via its nonenzymatic effects. Mature active MPO isolated from normal human neutrophils is a 145 kDa homodimer, which consists of 2 identical protomers, connected by a single disulfide bond. By binding to CD11b/CD18 integrin, dimeric MPO induces neutrophil activation and adhesion augmenting leukocyte accumulation at sites of inflammation. This study was performed to compare the potency of dimeric and monomeric MPO to elicit selected neutrophil responses. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. Analysis of the crucial signal transducer, intracellular Ca2+, showed that dimeric MPO induces Ca2+ mobilization from the intracellular calcium stores of neutrophils and influx of extracellular Ca2+ whereas the effect of monomeric MPO on Ca2+ increase in neutrophils was less. It was also shown that monomeric MPO was less efficient than dimeric MPO at inducing actin cytoskeleton reorganization, cell survival, and neutrophil degranulation. Furthermore, we have detected monomeric MPO in the blood plasma of patients with acute inflammation. Our data suggest that the decomposition of dimeric MPO into monomers can serve as a regulatory mechanism that controls MPO-dependent activation of neutrophils and reduces the proinflammatory effects of MPO.

Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase

CP is a copper-containing ferroxidase of blood plasma, which acts as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by MPO, such as HOCl, HOBr, and HOSCN, on its spectral, enzymatic, and anti-inflammatory properties was studied. We monitored the chemiluminescence of lucigenin and luminol along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by PMA or fMLP. Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr, and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced, along with a drop in the ferroxidase and amino oxidase activities of CP. Likewise, its inhibitory effect on the halogenating activity of MPO was diminished. Sera of either healthy donors or patients with Wilson disease were co-incubated with neutrophils from healthy volunteers. In these experiments, we observed an inverse relationship between the content of CP in sera and the rate of H2O2 production by activated neutrophils. In conclusion, CP is likely to play a role of an anti-inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO-mediated oxidative modifications.