Hypochlorous Acid-Modified Serum Albumin Causes NETosis in the Whole Blood Ex Vivo and in Isolated Neutrophils.

Type 2 diabetes mellitus (T2DM) is accompanied by halogenative stress resulting from the excessive activation of neutrophils and neutrophilic myeloperoxidase (MPO) generating highly reactive hypochlorous acid (HOCl). HOCl in blood plasma modifies serum albumin (Cl-HSA). We studied the formation of neutrophil extracellular traps (NETs) in the whole blood and by isolated neutrophils under the action of Cl-HSA. It was found that Cl-HSA induces neutrophil priming and NETosis. MPO-containing as well as MPO-free NETs were found. These NETs with different composition can be a product of NETosis of one and the same neutrophil. NET formation in neutrophils with vacuolated cytoplasm was detected. In the presence of Cl-HSA, acceleration of NET degradation was observed. Accelerated NET degradation and neutrophil priming can be the factors contributing to the development of complications in T2DM.

The Effect of Vitamin D3 on Neutrophil Extracellular Trap Formation in High-Glucose Conditions.

NETosis, i.e., the formation of neutrophil extracellular traps (NET), and neutrophil autophagy are important elements in the pathogenesis and the development of complications of type 2 diabetes mellitus (T2DM). Therefore, the search of drugs that can regulate the level of NETosis and autophagy in T2DM is relevant. Here we studied an ex vivo NET formation and neutrophil death in whole blood from healthy subjects upon the addition of glucose up to a high concentration of 15 mM or/and the phorbol ester PMA (phorbol-12-myristate-13-acetate). Their individual and combined action caused neutrophil death and an increase in NET content. It can be hypothesized that this resulted from activation of NETosis and autophagy. It was also shown that this activation of NETosis and autophagy is completely prevented by daily intake of 1000 IU vitamin D3 for 14 days. Therefore, vitamin D3 supplementation can be considered as a preventive measure against the development of T2DM complications.

Features of Ductular Reaction in Rats with Extrahepatic Cholestasis.

Ductular reaction develops during liver regeneration, fibrosis, and carcinogenesis. However, the types, stages of formation, and topography of ductular profiles in various pathologies remain insufficiently studied. Using the model of common bile duct occlusion, we showed that the number and topography of ductular profiles are closely related to the duration of biliary obstruction. The ductular profiles can be located inside the portal tract, along the existing bile ducts, and/or intramurally, around the portal vein, periportally, inside the lobules, in the portocaval fibrous connections, in the adventitia of the hepatic veins, in the septs connecting the portal tracts, and also in the "portal plate" of the liver. The ductular profiles can be formed as a result of expansion of existing bile ducts, cholangiocyte proliferation, as well as transdifferentiation of hepatocytes and activation of mesenchymal stem cells.

Spatial Organization of the Transport of Interstitial Fluid and Lymph in Rat Liver (Scanning Electron Microscopy of Injection Replicas).

Using the method of scanning electron microscopy of injection replicas, we studied the movement of a new injection mass between the blood microcirculation system, interstitial space, lymphatic system, and bile transport system in rat liver under normal conditions and 3 days after the occlusion of the common bile duct. The casts of the perisinusoidal spaces of Disse's after injection of the injection mass through the portal vein and common bile duct were obtained. Their direct transition not only in "leakages" structurally related to lymphatic capillaries in interlobular spaces, but also in perivascular spaces around the portal and hepatic veins. The flow of the injection mass through the perivascular spaces leads to the formation of peculiar "sheaths" around hepatic veins and components of the portal complex. The proposed approach allows effective visualization of the structural basis of interaction of various compartments of the fluid microcirculation in the liver under normal and pathological conditions.

Hyperglycemia and Some Aspects of Leukocyte Activation In Vitro.

We analyzed functional status of blood leukocytes in diabetes mellitus and after addition of glucose in vitro. To this end, generation of ROS and reactive halogen species by monocytes and neutrophils from patients with diabetes mellitus and healthy donors was assayed using lucigenin- and luminol-dependent chemiluminescence after stimulation with phorbol 12-myristate 13-acetate or opsonized zymosan in vitro. Formation of neutrophil extracellular traps was evaluated in the blood after addition of glucose. In comparison with donors, leukocytes from patients with diabetes mellitus were primed and this effect can be modeled by addition of glucose to the blood in vitro. Addition of glucose to donor blood also triggered the formation of neutrophil extracellular traps.

Investigation of the thermo stability of aluminum thin-film filters with protective MoSi2 cap layers.

Aluminum thin-film spectral filters are widely used in telescopes for space observations of the Sun in the extreme ultraviolet range of wavelengths. The main purpose of film filters is to block radiation in the UV, visible, and near-IR spectral ranges. In connection with the development of new projects for the observation of the Sun from close distances, the thermal stability of the entrance film filter is an important characteristic. In this paper, the thermal stability of Al films with 2.5 nm thick MoSi2 protective cap layers has been studied. MoSi2 was chosen as a coating material because MoSi2 caps effectively protect the Al film from oxidation and simultaneously increase the mechanical strength of the Al film. Vacuum annealing of MoSi2/Al/MoSi2 films was carried out at temperatures up to 300°C. It has been demonstrated that at an annealing temperature of more than 200°C for 24 h, a noticeable decrease in the blocking properties of the MoSi2/Al/MoSi2 film is observed in the visible wavelength range, which is caused by the appearance of semi-transparent crystalline silicon dendritic structures that are tens of micrometers in size in the film. In the annealed area of the film specimen, the intermetallic Al12Mo phase was detected by electron diffraction structure analysis, indicating a possible reason for the appearance of silicon atoms needed for dendrite growth as a result of the chemical interaction of Al and MoSi2. Due to the requirements for a high degree of visible radiation blocking (106 to 107 times), the appearance of even one dendritic structure significantly reduces the blocking properties of the film filter and is, therefore, not permissible. Based on the measurement of the transmission of MoSi2-2.5 nm/Al-72 nm/MoSi2-2.5 nm films at 633 nm for isothermal annealing at 200°C-250°C, the activation energy for the formation of dendritic structures (E=1.55±0.1 eV) was measured and the maximum permissible film temperature (130°C) at which dendritic structures did not appear during a 5-year mission was predicted.

Influence of barrier interlayers on the performance of Mo/Be multilayer mirrors for next-generation EUV lithography.

A comparative study was carried out of the structure and reflection performance of four types of multilayer mirror for extreme ultraviolet lithography at 11.2 nm; these were a pure Mo/Be structure and three Mo/Be-based structures with thin B4C, C and Si interlayers. It was demonstrated that Mo/Be mirrors show maximum reflectance at normal incidence, while maximum structural perfection is shown by Mo/Be/Si mirrors. The introduction of B4C and C layers into the structure increases the interlayer roughness and reduces the sharpness of the interfaces, adversely affecting the target coating characteristics. Results are presented for studies using four techniques: X-ray reflectometry, small-angle X-ray scattering, atomic force microscopy, and transmission electron microscopy.

Binding of Mucin by E. coli from Human Gut.

Cells of E. coli isolates from the gut of healthy volunteers (N=5) and patients with Crohn's disease (N=5) and laboratory E. coli strain DH5α bound mucin in vitro in similar amounts ranging from 0.02 to 0.12 mg/mg of bacterial dry weight. Binding was evaluated by the decrease in optical absorption of mucin solution at 214 nm after incubation with bacteria. Detailed analysis of mucin binding by one of isolates showed that during incubation of 0.09 mg/ml bacteria in 0.15 M NaCl containing 0.1 mg/ml mucin at 25oC, maximum binding was reached in 30 min, while in the presence of 14 mM α-methyl mannoside, mucin binding decreased by 46% (p<0.05). Confocal microscopy revealed intensive binding of FITC-labeled mucin to the surface of a small number of bacterial cells. Mucin binding did not significantly affect zeta potential of bacteria and their energetic status assessed by ATP content; at the same time, ATP content in the extracellular environment slightly increased.

A method for estimating magnetic field of TEM objective lens.

We propose a method for calibration of magnetic field in the objective lens of transmission electron microscope. The calibration process is based on classical Fresnel imaging of Permalloy disks and measuring the displacement of the magnetic vortex core when the sample is tilted at various excitations of the objective lens. We adopted the Carl Zeiss LIBRA 200 MC transmission electron microscope for Lorentz electron microscopy using this method. The objective lens magnetic field evaluation is tested on the Co/Pt multilayered films with known magnetic properties.

[Glutathione-dependent regulation of platelet aggregation with neutrophils and tumor cells].

It is shown that in the presence of reduced glutathione at low concentrations (1-5 microM) the extent of platelet aggregation with neutrophils increases and the lag period of platelet aggregation induced by tumor cells decreases. At the same time in the presence of reduced glutathione at high concentration (3 mM) the extent of platelet aggregation with neutrophils decreases, and the lag period of platelet aggregation induced by tumor cells increases. It is established that glutathione-dependent regulation of the intercellular contact formation between platelets and neutrophils depends on the ratio of glutathione oxidized and reduced forms: at fixed total glutathione concentration of 5 microM, increase of glutathione redox potential from -175 mV to 0 mV led to reduction in platelet aggregation with neutrophils. Thus, it is shown for the first time, that GSH has priming effect on the platelet aggregation with neutrophils and tumor cells, which may contribute to the regulation of inflammatory diseases and cancer.

Optical and magneto-optical resonances in nanocorrugated ferromagnetic films.

We report here on optical and magneto-optical spectra investigations of nanostructured Co and Ni films deposited on top of polymethyl methacrylate (PMMA) colloidal crystal. Resonant peculiarities have been observed in both spectra; their positions are scaled with the PMMA sphere diameter and depend on incident angle. Asymmetry of the resonance lineshapes as well as change of the direction of magneto-optical rotation has been observed. Both the surface plasmon resonance and the interference between reflections from the colloidal crystal and from the nanostructured film should be considered to explain the obtained results.

[Neutrophils as a source of factors that increase the length of the inflammatory phase of wound healing in patients with type 2 diabetes mellitus]. / Neitrofily kak istochnik faktorov, uvelichivaiushchikh prodolzhitel'nost' fazy vospaleniia ranevogo protsessa u bol'nykh sakharnym diabetom 2-go tipa.

Oxidative stress and neutrophil activation leading to an increase in myeloperoxidase (MPO), elastase and neutrophil extracellular trap (NET) levels in blood are considered as pathogenic mechanisms responsible for the development of extremity damage in people with type 2 diabetes mellitus (T2DM). The aim of this study was to analyze the relationship between factors, associated with neutrophil activation, and the length of the initial phase of wound healing (the inflammatory phase) in T2DM patients. Patients were divided retrospectively into three groups depending on the damage extent: group 1 (wound on toe) < group 2 (wound on foot) < group 3 (wound on lower leg). Compared to the control group (healthy volunteers), T2DM patients at admission to hospital had significantly (p<0.05) increased levels of blood glucose and glycated hemoglobin (groups 1-3), ESR (groups 1 and 3), blood neutrophil count (groups 2 and 3), plasma MPO concentration (groups 1-3) and blood NET concentration (group 3) and decreased levels of plasma thiols (groups 1-3) and erythrocyte glutathione peroxidase activity (groups 2 and 3). The length of hospital stay after surgical procedures corresponded to the length of the inflammatory phase of the wound healing process and correlated with the number of blood neutrophils in patients before surgery (r=0.72, p<0.05). Leukocytic intoxication index depended on wound area (r=0.59, p<0.05), and it was significantly higher for groups 2 and 3 compared to the control group and group 1. The neutrophil count before surgery in T2DM patients with damage in the lower extremities correlated with the length of the inflammatory phase of wound healing. The correlation found can be attributed to an increase in extracellular MPO and NETs, which, in its turn, results from the activation and degranulation of neutrophils and netosis. Thus, the duration of the inflammatory phase of wound healing depends on specific aspects of systemic inflammation increasing oxidative/halogenative stress and intoxication.

[Effects of infusion media on human red blood cell morphology].

The effect of various infusion media on the structure of human red blood cells was evaluated in vitro and in vivo. The in vitro experiments used 10% sodium chloride (NaCl) solution, 10% glucose solution, 20% albumin solution, Rheopolyglucin, HyperHAES solution (18 g of NaCl in combination with 60 g of hydroxyethylstarch (HES), 200/0.5), Voluven (HES 130/0.4/9:1), and a combination of hypertensive NaCl solution and Rheopolyglucin. The morphofunctional response of red blood cells was studied in the clinical setting when 6% Voluven solution (HES 130/0.4/ 9:1) and hypertensive NaCl and glucose solutions were used. It was established that 10% NaCl solution caused considerable changes in the morphology of red blood cells both in the experiment and in patients with severe brain injury. The magnitude of structural changes increased as blood NaCl concentrations became higher. 10% glucose solution, Voluven, Rheopolyglucin, and albumin did not virtually affect the structure of red blood cells. Infusion of Voluven (500 ml of 6% solution for 40 minutes) induced no changes in the morphology of red blood cells in the clinical setting. Among the test solutions used to correct intracranial hypertension (HyperHAES, 10% NaCl, a combination of rheopolyglucin and 10% NaCl), HyperHAES exerted the least effect on the morphology of red blood cells.

Photoinduced formation of thiols in human hair.

Raman, scanning electron, and optical microscopy of hair and spectrophotometry of soluble hair proteins are used to study the effect of UV-vis radiation on white hair. The samples of a healthy subject are irradiated using a mercury lamp and compared with non-irradiated (control) hair. The cuticle damage with partial exfoliation is revealed with the aid of SEM and optical microscopy of semifine sections. Gel filtration chromatography shows that the molecular weight of soluble proteins ranges from 5 to 7kDa. Absorption spectroscopy proves an increase in amount of thiols in a heavier fraction of the soluble proteins of irradiated samples under study. Raman data indicate a decrease in the amount of SS and CS bonds in cystines and an increase in the amount of SH bonds due to irradiation. Such changes are more pronounced in peripheral regions of hair. Conformational changes of hair keratins presumably related to the cleavage of disulfide bonds, follow from variations in amide I and low-frequency Raman bands. An increase in the content of thiols in proteins revealed by both photometric data on soluble proteins and Raman microspectroscopy of hair cuts can be used to develop a protocol of the analysis of photoinduced hair modification.

Cytochrome P450 database.

This paper describes a specialized database dedicated exclusively to the cytochrome P450 superfamily. The system provides the impression of superfamily's nomenclature and describes structure and function of different P450 enzymes. Information on P450-catalyzed reactions, substrate preferences, peculiarities of induction and inhibition is available through the database management system. Also the source genes and appropriate translated proteins can be retrieved together with corresponding literature references. Developed programming solution provides the flexible interface for browsing, searching, grouping and reporting the information. Local version of database manager and required data files are distributed on a compact disk. Besides, there is a network version of the software available on Internet. The network version implies the original mechanism, which is useful for the permanent online extension of the data scope.

[Characteristics of the ultrastructural organization of endothelial cells in cardiac and omental blood capillaries based on cryofractographic data]. / Nekotorye osobennosti ul'trastrukturnoi organizatsii éndotelial'nykh kletok krovenosnykh kapilliarov serdtsa i sal'nika po dannym metoda zamorazhivaniia-skalyvaniia.

Using cryofractography, endotheliocytes of heart and omentum blood capillaries were studied, different patterns of distribution of membranous particles being observed of the "P" and "E" faces of the fracture plans of endothelial cell cytomembranes. Three types of distribution of micropinocytotic vesicles can be distinguished: the linear, hexagonal, and the clusteral type. The number of micropinocytotic vesicles was sharply reduced near the region of intercellular contacts. On the fracture plan, attached pinocytotic vesicles look like "craters" on the "E" face, and like circumvallatue papillae on the "P" face. Cell membranes, making intercellular contacts, have a network structure consisting of ridges on the "P" face and of grooves on the "E" face. Such a molecular membrane organization is discussed from the standpoint of modern concepts of the receptor--cytosceletal complex. It is supposed that this complex may control processes of transendothelial mass--transfer.

[The detection of the cholesterol of the erythrocyte plasmalemma by using a latex marker]. / Vyiavlenie kholesterina plazmalemmy éritrotsitov s pomoshch'iu lateksnogo markera.

Using a new specific cholesterol marker (SCM) and scanning electron microscopy, the distribution of cholesterol in the plasmalemma of rabbit and human erythrocytes was studied. Investigation of SCM linking with the erythrocyte membranes with different cholesterol/phospholipid indices shows that the increase of cholesterol/phospholipid index of the erythrocyte membrane correlates with the increase in the number of SCM particles on the erythrocyte surface.

[A quantitative analysis of the regional organizational characteristics of the aortic endotheliocyte layer in normotensive and spontaneously hypertensive rats]. / Kolichestvennyi analiz regionarnykh osobennostei organizatsii plasta éndoteliotsitov aorty normotenzivnykh i spontanno gipertenzivnykh krys.

Using scanning electron microscopy and semiautomatic image analyser, regularities and quantitative changes were studied in endotheliocytes in three regions of thoracic aorta during ageing of normal and spontaneously hypertensive rats of the Kyoto-Wistar line. The data of morphometric analysis and the results of simulating endothelial cell monolayer changes, by means of construction of polyrow mosaics, suggest that the number of small endotheliocytes of normotensive rats in all aorta regions is lowered in the period from 2 to 10 months to increase then from 10 to 16 months. The endothelial cell monolayer changes occur as follows: the small endotheliocytes appear and then disappear, keeping their random distribution in the endothelial layer. Within the same period, in spontaneously hypertensive rats, the behavior of the endothelial layer in the region of intercostal arteria ostia and on the dorsal surface is similar to that in normal rats, being presumably associated with age changes. In hypertensive rats, on the ventral surface of the aorta, a relative number of small cells increases at the expense of small cell foci formation. Thus, the provided evidence points to a heterogeneity of responses of endothelial layers in different regions of the aorta on the constant elevation of pressure.

[Detection of the intercellular borders of the endothelium using scanning electron microscopy]. / Vyiavlenie mezhkletochnykh granits éndoteliia s pomoshch'iu skaniruiushchei élektronnoi mikroskopii.

Three different approaches for studying aorta endothelial cells by scanning electron microscopy (SEM) were compared to expose cell border lines and surface shape simultaneously. Staining of aorta probes with silver nitrate leads to more specific silver deposits at cell junctions, but unsatisfactory image resolution obscures any detail of cell junctions and other fine structural features. Endothelium impregnated with tannin-osmium showed enhanced cell junction detail, but this method resulted in formation of presumably artificial structures. Osmium tetraoxide was most optimal for impregnation of aorta probes, as it exposed well cell border lines, excluded artifacts of tannin-osmium staining, provided a good image resolution in SEM, and allowed to analyse the ultrastructural features of cell surface.

[The distribution of negative charges on the luminal surface of Descemet's endothelium]. / Raspredelenie otritsatel'nykh zariadov na liuminal'noi poverkhnosti destsemetova éndoteliia.

The localization of anionic sites on the surface of corneal endothelium was investigated using colloidal nickel. The distribution of the binding sites of colloidal nickel along the cell surface was examined with the scanning electron microscope. The application a special detector of high sensitivity reflection allows to register a composed contrast determined by colloidal nickel. Colloidal nickel is bound with the surface of endothelium in areas of cell junctions only. A 1 X 10(-4)% sol. adrenaline injection into the anterior rabbit eye chamber in vivo changed the distribution of colloidal nickel. These findings demonstrate that colloidal nickel allows to mark the anionic sites on the cell surface for scanning electron microscopic studies.