Neutrophils are the leading cells of the innate immune system and the main population of leukocytes responsible for the primary reaction of the body to various infectious particles. The latter are destroyed by neutrophils due to the processes of phagocytosis and a cascade of reactions, including a respiratory burst reaction. As a result of the respiratory burst reaction of neutrophils they produce reactive oxygen and halogen species, powerful cytotoxic agents that destroy particles in the phagolysosome. All of these processes require regulation, because overactivation of neutrophils can lead to the damage of the tissues surrounding the focus of inflammation mediated by the reactive forms of oxygen. Proteins of the acute phase of inflammation claim for the role of inflammatory regulators. Previously, we have shown the participation of ceruloplasmin in inhibiting the respiratory burst reaction of neutrophils in blood samples. Fibrinogen, on the contrary, increased the intensity of the respiratory burst reaction. Not for all proteins of the acute phase of inflammation and especially their combinations, the effect on the functions of neutrophils has been studied in detail. In this work the effect of a number of proteins of the acute phase of inflammation (C-reactive protein, serum amyloid A, alpha-1-acid glycoprotein and fibrinogen) on the ability of peripheral blood neutrophils to the respiratory burst reaction was studied for the first time using flow cytometry with the registration of the production of reactive oxygen species in cells of peripheral blood samples. Significant changes in the ability of neutrophils to produce reactive oxygen species were found for a number of combinations of the studied proteins of the acute phase of inflammation. The study of interactions of ceruloplasmin and fibrinogen with peripheral blood neutrophils revealed their membrane localization. It seems promising to identify receptors for proteins of the acute phase of inflammation on the neutrophil membrane.

The HeLa cell’s nuclei surface landscape, which appeared after the mechanical deformation, was studied by atomic-force microscopy. Nuclei were isolated from HeLa-line cells, and deformed under centrifugal forces, followed by fixation with glutaraldehyde. The resulting landscape was shown to be mainly due to the high chromatin resistance to deformation. This stability correlates with DNA supercoiling. Cells’ treatment with topoisomerase inhibitors I and II leads to the relaxation of supercoiling and significant flattening of the nuclei. On the contrary, cells’ treatment with a DNA intercalator leads to an increase in the DNA rigidity and, as a result, the resistance of chromatin to mechanical deformation. Thus, the observed changes in morphology reflect the functional features of the cell nucleus.

Stochastic gene expression can play an important role in the fate of both each specific cell and the cell population as a whole. In this work, we demonstrate significant variability of p53 expression, up to its complete absence, in human primary glioblastoma cells, which is not observed in cells of normal FLEH fibroblasts and transferable tumour cell lines, such as HeLa and HT1080. At the same time, the variability of p53 expression in cells depends on the density of seeding. We show that when the transcription is suppressed with actinomycin D or the p53 gene is knocked down by RNA interference, the restoration of its expression level in cells proceeds stochastically.

Background: Nuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. Methods: Isolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM). Results: Nuclei of the normal fibroblast cells were completely flattened under mechanical stress, whereas nuclei of the cancerous HeLa were extremely resistant. In the deformed HeLa nuclei, AFM revealed a highly-branched landscape assembled of similar to 400 nm closed-packed globules and their structure was changing in response to external influence. Normal and cancerous cells' isolated nuclei were strikingly different by DNA resistance to applied mechanical stress. Paradoxically, more transcriptionally active and less optically dense chromatin of the nuclei of the cancerous cells demonstrated higher physical rigidity. A high concentration of the transcription inhibitor actinomycin D led to complete flattening of HeLa nuclei, that might be related to the relaxation of supercoiled DNA tending to deformation. At a low concentration of actinomycin D, we observed the intermediary formation of stochastically distributed nanoloops and nanofilaments with different shapes but constant width similar to 180 nm. We related this phenomenon with partial DNA relaxation, while non-relaxed DNA still remained rigid. Conclusions: The resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM. General significance: A new outlook to studying internal nuclei structure is proposed.

The small-angle neutron scattering (SANS) on HeLa nuclei demonstrates the bifractal nature of the chromatin structural organization. The border line between two fractal structures is detected as a crossover point at Qc approximate to 4x10-2 nm-1 in the momentum transfer dependence Q-D. The use of contrast variation (D2O-H2O) in SANS measurements reveals clear similarity in the large scale structural organizations of nucleic acids (NA) and proteins. Both NA and protein structures have a mass fractal arrangement with the fractal dimension of D approximate to 2.5 at scales smaller than 150 nm down to 20 nm. Both NA and proteins show a logarithmic fractal behavior with D approximate to 3 at scales larger than 150 nm up to 6000 nm. The combined analysis of the SANS and atomic force microscopy data allows one to conclude that chromatin and its constitutes (DNA and proteins) are characterized as soft, densely packed, logarithmic fractals on the large scale and as rigid, loosely packed, mass fractals on the smaller scale. The comparison of the partial cross sections from NA and proteins with one from chromatin as a whole demonstrates spatial correlation of two chromatin's components in the range up to 900 nm. Thus chromatin in HeLa nuclei is built as the unified structure of the NA and proteins entwined through each other. Correlation between two components is lost upon scale increases toward 6000 nm. The structural features at the large scale, probably, provide nuclei with the flexibility and chromatin-free space to build supercorrelations on the distance of 103 nm resembling cycle cell activity, such as an appearance of nucleoli and a DNA replication.

Despite the introduction of new anti-tuberculosis drugs, the problem of treating tuberculosis patients with a broad drug-resistant pathogen (XDR MBT) remains relevant.Aim of the study. Тo increase the effectiveness of treatment of this category of patients using an autologous composition based on dendritic cells activated by a specific antigen.Research materials and methods. А prospective study conducted in 2016-2019 included 52 patients, 36 (69.2%) men and 16 (30.8%) women, average age 43.2 ± 1.5 years. All patients received antituberculosis therapy in accordance with existing regulatory documents and recommendations. The main group consisted of 25 patients (48.1%) who agreed to immunotherapy with the introduction of an autologous composition with activated specific peptides ESAT-6 and SFP-10 dendritic cells against the background of anti-tuberculosis therapy. Statistical processing of the material carried out using the Statistica 10.0 application software package.Results. In 70.0% of cases (n = 14), there were no adverse reactions to suspension administration in patients of the main group. One patient (7.2%) had a general reaction in the form of tremor, chills after the first injection. In 5 (38.7%) patients on the 8th and 9th administration, a skin reaction was noted at the injection sites in the form of a papule, with a final regression of these changes after 8 weeks. By 18– 24 months of the course, anti-tuberculosis therapy in combination with immunotherapy in the first month of treatment allowed to obtain a positive effect according to clinical, bacteriological and radiological data in 64% of cases. It was not possible to obtain this result during therapy at the previous stages of treatment. In the comparison group, a positive result achieved only in 45.5% of cases.Conclusion. The results of the study suggest a positive effect of complex treatment with the inclusion of immunotherapy with an autologous composition based on dendritic cells in the most severe contingent of patients with pulmonary tuberculosis with broad drug resistance of the pathogen, even with an ineffective course of chemotherapy at the previous stage in 64% of cases.

The etiology of sarcoidosis is not completely understood. A hypothesis exists about the relationship between sarcoidosis and a complex of pathological autoimmune reactions that occur under the influence of triggering factors. In this study, specific immune complexes in the blood plasma of patients have been determined, which can indirectly reveal the causes of the disease.The study included 33 patients with lung sarcoidosis (I group), compared to 24 healthy donors who served as a control group (II group). The patients underwent standard examination. Their blood plasma was investigated by the dynamic light scattering method with addition of tuberculosis antigens (ESAT-6/SFP-10) and “lung healthy tissue extract”. Statistical analysis was performed using the Statistica 7.0 program. Test results were considered significant at p < 0.05.Аccording to the data obtained, addition of ESAT-6/SFP-10 to patient’s blood plasma almost did not lead to the formation of immune complexes in most samples. Meanwhile, development of such complexes after addition of “lung tissue extract” was revealed in all the patients. The immune complexes were not detected in any donor from control group after stimulation with both kinds of antigens (p < 0.01).The data on distinct formation of immune complexes with the addition of “lung healthy tissue extract” in patients with lung sarcoidosis may be considered an indirect evidence for occurrence of autoimmune reaction under the influence of some pathogenic factors. Absence of de novo immune complex formation after addition of tuberculosis antigens (ESAT-6/SFP-10) makes it unlikely any direct effects of tuberculosis bacteria upon development of sarcoidosis.

The diagnostic of tuberculosis infection, including the use of immunological methods, evolved significant changes. The introduction of new diagnostic tests allowed to improve the diagnosis of latent tuberculosis infection (LTI). However, the positive results of immunological tests in both tuberculosis patients and in those with LTI do not allow to divide these conditions, which requires the development and implementation of new diagnostic approaches.Materials and methods. A prospective study with a survey of two groups of patients was conducted: group I (n=50) - patients with verified pulmonary tuberculosis, MBT (+); group II (n =15) – subjects with LTI and control group – healthy subjects (n=14). The examination complex included clinical, radiological, bacteriological, immunological (Mantoux test with 2 TU, T-SPOT.TB, QFT and Diaskintest) methods. Immune complexes were determined in all patients and healthy individuals by the method of dynamic light scattering after the in vitro addition of specific antigens - peptides ESAT-6 and SFP-10.Results. The obtained data demonstrate the low informativeness of the clinical method in the diagnostic of pulmonary tuberculosis. In the presence of characteristic X-ray changes, bacteriological verification of tuberculosis was obtained only in 46% of cases. The use of various immunological tests allows to obtain positive test results in 84-90% of cases simultaneously with the 100% of the positive results in subjects with LTI. Determination of specific immune complexes by the method of dynamic light scattering allows to determine the activity of tuberculosis infection in 100 % of cases and to identify a high-risk group for the development of active tuberculosis in people with latent tuberculosis infection.Conclusions: the obtained data can be applied not only in the diagnosis of active tuberculosis in the absence of diagnosis verification, but also allow to identify a high-risk group for the development of the disease in people with latent tuberculosis infection.

Early diagnosis of tuberculosis remains essential for the prevention of the disease and control of its incidence. The diagnosis of latent tuberculosis infection (LTBI) has significantly improved over the last years due to the use of new immunological tests, including the Diaskintest.The aim of the study was to assess the potential of immunological assays for diagnosis of latent tuberculosis infection and determination of risk groups for tuberculosis among personnel of different institutions.Materials and methods: in 2015–2018, a prospective group-control study was conducted that included employees of various medical centres (n = 154) and healthy individuals (n = 30). There were no significant age differences between the subjects. All the subjects included in the study shared their medical history, underwent general examination, X-ray examination, and were tested with Diaskintest. Multispiral computed tomography (MSCT) of the chest was performed for every subject with a positive Diaskintest result.Results: the comparison of the number of persons with LTBI in general care hospitals, personnel of a manufacturing firm, and among healthy individuals showed that the risk of being infected with Mycobacterium tuberculosis is very similar in all the groups (from 6.8 to 10.0 %). The highest risk of developing LTBI was found in people who worked in an infectious area for more than 5 years (48.4 %). The analysis of the level of immune complexes (ICs) demonstrated that individuals with LTBI did not have isotypes of IgG3 and IgE immune complexes, which are found in patients with tuberculosis. The identification of particular isotypes indicates a borderline state between LTBI and tuberculosis and requires therapeutic intervention.Conclusions: the use of the Diaskintest makes it possible to distinguish groups of employees of various institutions who are at a higher risk of developing tuberculosis. At the same time, determination of the ICs helps to reveal the borderline condition which requires medical intervention.

In this study, we present a model for visualization of the exosome transfer of p53-GFP protein between cultured mammalian cells. The temporal parameters of the accumulation of fluorescent labeled protein in recipient cells have been analyzed. We employed HEK293 cells transfected with p53∆Y126-GFP plasmid (a GFP clone) as donor of the exosomes, while the original HEK293 cell line was used as a recipient. Our results provide evidence that the transfer of the GFP protein from cell to cell is carried out via exosomes. It has been shown that the accumulation of this protein by the recipient cells in cocultures of these cells takes a prolonged time. The temporal parameters of this transfer differ between the cells within the same population.

Properties and mechanisms of PCNA (proliferating cell nuclear antigen) functions have been investigated for a long time and are studied in great detail. As follows from its name, most known PCNA functions (DNA replication, DNA repair, DNA recombination and others) are connected with cell proliferation and localization of this protein in nuclei. In addition, there is good reason to believe that PCNA also performs some functions in the cytoplasm. However, the possible role and mechanisms of PCNA action in the cytoplasm require careful study and clarification. Interestingly, such cells as neutrophils differ in that they are non-dividing on one hand and on the other hand contain a rather large amount of PCNA, which is localized only in the cytoplasm, that is, they are an ideal model for the study of cytoplasmic PCNA. Using cross-linkages with formaldehyde, we showed that this cytoplasmic PCNA is cross-linked in a similar way, that is, organized in the same way as the nuclear PCNA that is present in the proliferating cells. Previously, we showed that PCNA in such cells is organized into a dynamic complex of double trimer on the basis of the back-toback principle. Apparently, such organization of this hub-protein allows it to better coordinate the processes taking place in the cytoplasm as well.

Glioblastomas are tumors of neuroectodermal origin with a high degree of diversity. Tumor cells isolated from the surgical material of patients with glioblastomas are heterogeneous populations varying in morphology, phenotype, and genetic characteristics. This paper presents a description of two new glioblastoma cell lines, R1 and T2, isolated from tumor tissue of patients in 2010. We investigated their morphological and cytochemical cell characteristics and the expression of neuronal, mesenchymal, and endothelial markers, as well as the activity of genes encoding a number of growth factors, extracellular matrix proteins, and intracellular proteins typical for cells of mesenchymal origin. R1 and T2 cell lines are morphologically different. T2 cell line was characterized by the presence of multinuclear cells. In terms of the expression of β-tubulin III, MGMT, and p53 protein, R1 cell line was more heterogeneous than T2. R1 and T2 glioblastoma cell lines also differed in the presence and ratio of cell populations with mesenchymal, neuronal, and endothelial markers. Thus, neuronal markers CD133/2 and CD56 were detected only on R1 cells. Both lines were characterized by high activity of growth factor genes TGFβ1, VEGF , and FGF2 ( b ), lower activity of EGF , and high expression of THBS1 and αSMA genes. However, the activity of most of the genes under study in R1 cells was higher than in T2 cells. The greatest difference lay in the expression of HGF, FAP , and TNC . Comparison of two new glioblastoma cell lines, R1 and T2, with the continuously cultivated lines А172 and T98G showed that R1 line had remarkable similarity with A172, while T2 glioblastoma resembled T98G cells. Apparently, the differences between R1 and T2 cell lines are determined by the properties of the initial tumors rather than the time of the cell cultivation.

In this paper we introduce new approach in formed in heterogeneous biological fluids immune complexes analysis. The approach is based on an analysis of the contribution of immune complexes in dynamic light scattering, recorded by laser correlometer with heterodyne measurement circuit. We demonstrated our approach sensitivity using embryonic calf serum model in addition of monoclonal antibody against hepatocuprein. Proposed approach is capable to detect forming immune complexes by adding antigen at a concentration greater than 150 pg/ml. Using immunoaffinity techniques we confirmed the lawfulness of identified peaks in the histograms of dynamic light scattering as appropriate immune complexes. For these purposes, we selectively removed immune complexes using staphylococcal protein A sepharose immobilized on the particles or increased in size immune complexes due to their further aggregation using antibodies specific for human immunoglobulin. The proposed approach can detect immune complexes in various biological fluids.

Exosomes are nanosized vesicles that are secreted by many types of cells. We have found that exosomes secreted by HEK293 and HT-1080 can suppress growth and proliferation of p53-deficient cells. Upon overexpression of exogenous p53-GFP in HEK293 cells, we observed p53 protein in exosomes that were secreted by these cells. We also found endogenous p53 in exosomes that were secreted by HT-1080 cells with a higher level of p53 expression. We were able to detect endogenous p53 protein in exosomes that originated from human plasma and were transferred to p53-deficient cells. Our findings indicate that p53 protein can be transferred between cells and may play an important physiological role.

Clinical and radiological features of tuberculosis and sarcoidosis are quite overlapping, and therefore, a diagnostic dilemma often persists. There are no commonly accepted criteria for the diagnosis of sarcoidosis due to the lack of data on the etiology of the disease. The exclusion of tuberculosis in every patient with suspected sarcoidosis is a mandatory stage of diagnosis, especially in countries with a high burden of tuberculosis. A prospective study was conducted with two groups of patients: group I (n = 50)—patients with pulmonary sarcoidosis established according to standard criteria; group II (n = 28)—patients with pulmonary tuberculosis with bacterial excretion. The control group (n = 24) was presented by healthy subjects. The examination complex included x-ray, bacteriological, immunological (Mantoux test with 2 TE, TB.SPOT test), and histological methods. All patients and healthy subjects were assessed for immune complexes with the use of the dynamic light scattering (DLS) method and adding of “healthy lung tissue extract” antigens and specific tuberculosis antigens ESAT-6 and SFP-10 in vitro. Significant differences were found in determining specific immune complexes in patients with pulmonary sarcoidosis and pulmonary tuberculosis. Registration of specific immune complex formation with “healthy lung tissue extract” in 100% cases may indicate the autoimmune nature of sarcoidosis. The absence of the immune complex formation in response to ESAT-6/SFP-10 antigens can be used for the differential diagnosis of two diseases. The diagnostic significance of the DLS method was 100% for sarcoidosis and 92.2% for tuberculosis. The data obtained in the study allows not only understanding the etiology of sarcoidosis, but also obtaining new criteria for the differential diagnosis of tuberculosis and pulmonary sarcoidosis.

OBJECTIVE. Assessment of diagnostic significance of exosomal microRNAs analysis in colorectal cancer (CRC). MATERIALS AND METHODS. Plasma samples from 100 patients with colon tumors and 20 healthy donors. Exosomes were isolated by differential ultracentrif ugation the analysis was performed by dynamic light scattering (DLS), cryo-EM, flow cytometry. Quantitative analysis of exosomal microRNAs was performed by RT-PCR. To evaluate the results obtained, the Kraskel-Wallis statistical test and ROC analysis were used. RESULTS. It is shown that CRC causes characteristic changes in the concentration of a number of exosomal microRNAs. Analysis of «reciprocal miRNAs pairs» was proposed as algorithmfor personalized diagnostic of CRC. The optimal parameters of diagnostic values were obtained for miRNA pair«miR-223 / miR-181a» (sensitivity =0.93, specificity =0.88). CONCLUSION. The analysis of exosomal microRNAs presents a promising method for early diagnostics / screening of colorectal cancer.

Background. Multiple myeloma (MM) is a hematologic malignancy of plasma cells. The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of exosomes (EXs). The role that EXs, released by MM cells have in cell-to-cell communication and signaling in the bone marrow is currently unknown. EXs as a source of markers for MM diagnostics are also not studied.Objective: to use proteomic profiling of EXs as a tool to identify circulating tumor associated markers in MM patients.Results. The proteome composition of EXs obtained from plasma of patients with MM and multiple sclerosis was studied for the first time. nano-HPLC–MS/MS analysis identified a total of 332 proteins in the EXs of both groups of patients and determined the proximity of their qualitative composition. For the first time, 12 differentially expressed proteins were detected, the levels of which were significantly increased in EXs from patients with MM. This allowed us to consider them as potential markers of the disease.Conclusion. Proteomic analysis of EXs obtained from plasma of patients with MM is an important method for finding disease markers.

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.