Comparison of modulation interference microscopy, DNA spectrometry, DNA cytometry, and flow cytofluorimetry in the assessment of phytohemagglutinin-induced activity of human blood lymphocytes

Rationale: The study of the structural particulars and functional state of immune cells and primarily lymphocytes is of great importance for both fundamental and clinical medicine. It requires the development of simple and reliable analytic methods that would allow for fast and effective real-time assessment of cell activity.Aim: To evaluate the effectiveness of the interference microscopy compared to DNA spectrometry, DNA cytometry, and flow cytometry with an internalized fluorescent label CFSE (carboxyfluorescein succinimidyl ester) in the assessment of PHA-induced proliferation of human blood lymphocytes.Materials and methods: Phytohemagglutinin (PHA)-induced proliferative activity of blood lymphocytes from 10 healthy volunteers was studied with various methodological strategies. Blast transformation of lymphocytes was induced by their incubation for 5 days with PHA 5 μg/mL. The cell proliferative activity was assessed as follows: 1) by DNA spectrometry at 260/280 nm using Tecan Infinite 200 Pro with a specialized NanoQuant Plate™; 2) by cytophotometry followed by cell distribution analysis assessing deoxyribonucleic acid (DNA) content after staining with Felgen's dye with an imaging system based on an Olympus BX41 light microscope with a ProgRes CF camera; 3) by flow cytometry using an internalized fluorescent label CFSE; the analysis was performed with a BD FACS Calibur flow cytometer; 4) by measurement of the lymphocyte interference profile with a modulation interference microscope MIM-340 (Schwabe, Russia). The functional activity of the nucleus (FAN) was determined and used as a criterion for assessment of the lymphocyte functional state.Results: Incubation of lymphocytes with PHA led to an increase in the linear size by 22.2±2.8%, a decrease in phase height by 46.3±4.7% (p=0.019), and an increase in FAN by 75.9±9.4%, vs control (p=0.046). As measured by isolated DNA spectroscopy, PHA stimulation of lymphocytes was associated with an increase in the amount of DNA by 55% vs baseline (409.8±22.3 ng/μL and 264.3±25.0 ng/μL, respectively, p=0.049). Felgen's reaction revealed that the proportion of nuclei containing more than 2n DNA was 2% in the control cells and 14.8% in the PHA-activated lymphocytes, with a difference between the groups of 12.8%. CFSE staining with subsequent incubation and assessment by flow cytofluorimetry demonstrated an increase in the percentage of proliferating cells from 1.68±0.9% in the control to 55.56±5.6% (p=0.00068) in the mitogen-stimulated sample.Conclusion: Modulation interference microscopy does not require the sample preparation and demonstrated comparable and even higher effectiveness compared to conventional methods for assessment of lymphocyte activity. At the same time, it allows for evaluation of the lymphocyte functional state in real time in the process of cultivation. This opens ample opportunities for evaluation immune cells for research and diagnostic purposes.

Immunopathogenetic features and prognostic criteria for severe hemorrhagic fever with renal syndrome

Aim. Assessment of the features of cellular immunological mechanisms at the early stage of HFRS of varying severity and development of prognostic criteria for the risk of a severe course of the infectious process. Materials and methods. An immunological blood test (flow cytofluorimetry method) was performed in 12 patients with severe HFRS and 53 patients with moderate course in the dynamics of the disease. Statistical data processing was performed based on the SPSS software package. Results. At the initial stages of HFRS, immunological features of the severe course of the disease were established in the form of a higher content of T-helper and regulatory T-cells in the blood and a reduced number of CTL, including their activated pool. Based on these changes, an immunological prognostic coefficient of HFRS was developed, which allows determining the risk of severe course in the early days (febrile period) of the disease with high prognostic accuracy. Conclusion. The results obtained allowed us to identify previously unknown features of the immune process at the initial stages of HFRS development, which allowed us to propose a new approach to predicting the severe course of the disease.

Dynamics of Mitochondrial Transmembrane Potential Changes in Blood Monocytes in Conditions of Development and Course of Experimental Periodontitis and the Effect of Korvityn on it

Inflammatory diseases of periodontal tissues remain one of the most complex and unresolved problems of modern dentistry. The most important internal stimulus for triggering apoptosis is DNA damage in response to various factors (including reactive oxygen species). Mitochondrial transmembrane potential (Δψm) is generated by the electrochemical gradient of protons on both sides of the membrane and is closely related to the functioning of mitochondria, its support is provided by the processes of electron transfer in the respiratory chain. The purpose of our study was to elucidate the pathogenetic role of changes in mitochondrial transmebranic potential in the dynamics of the inflammatory response in experimental bacterial-immune periodontitis and the effects of quercetin (Korvityn) on it. Material and methods. The study was performed on white clinically healthy rats. Experimental bacterial-immune periodontitis in experimental animals was induced by injection of a mixture of microorganisms diluted with egg protein into the tissues of the periodontal complex. Quercetin was administered by intramuscular injection for correction. Evaluation of changes in mitochondrial transmembrane potential of leukocytes was performed by flow cytofluorimetry. Results and discussion. In experimental bacterial-immune periodontitis, the percentage of cells with reduced mitochondrial transmembrane potential among blood monocytes significantly increased. In animals on the 7th day of the study, the number of cells with reduced mitochondrial transmembrane potential among blood monocytes increased significantly compared with the control group. For the next study period (14th day), the number of cells with reduced ∆ψm decreased compared to the 7th day of the experiment. Having analyzed the data of mitochondrial transmembrane potential of blood monocytes on the 30th day of the experiment, we noted that they decreased relative to those obtained on the 14th day of the study, indicating profound oxidative imbalance in cells and destabilization of the mitochondrial membrane. The use of quercetin led to a decrease in the values compared to the data of animals with our simulated pathology on the 14th day, the experiment without the introduction of flavonol, but they remained significantly higher than the control group of animals. Conclusion. Flavonol (Korvityn) quercetin reduced mitochondrial transmembrane potential in experimental bacterial-immune periodontitis, which was evidence by stabilization and attenuation of the inflammatory process

Studies on correlations between immunophenotype and the indices of metabolic enzyme activity of blood lymphocytes in children with hypertrophy of the pharyngeal tonsils

The objective of our study was to evaluate correlation between the immune pheno-type and activity indices of NAD (P)-dependent dehydrogenases in peripheral blood lymphocytes in young children with hypertrophy of the pharyngeal tonsil (HPT). We have examined 57 children, 1-3 years of age, with hypertrophy of the pharyngeal tonsils (HPT). The control group consisted of 35 healthy children of the same age. The numbers of CD3+, CD4+, CD8+, CD19+, CD16+/56+ lymphoid cells in peripheral blood were determined by flow cytofluorimetry technique. Activity of NAD (P)-dependant dehydrogenases in peripheral blood lymphocytes was studied using bioluminescent method as described elsewhere (А. Savchenko, L. Suntsova, 1989). Correlation analysis has revealed an increase of positive correlations, a decrease of the correlation strength, and emergence of new connections between phenotype and activity indices of NAD (P)-dependent dehydrogenases in peripheral blood lymphocytes in children with hypertrophy of pharyngeal tonsils (HPT). Specific correlation patterns between the phenotype and activity indices of NAD (P)-dependent dehydrogenases in peripheral blood lymphocytes have been revealed in children with hypertrophy of pharyngeal tonsils (HPT).

PRURITUS AS A MARKER OF IMMUNE DISTURBANCES IN PSORIASIS

Objective: Based on the study of some indicators of cellular and humoral immunity, evaluate the features of immunoreactivity in patients with psoriasis with concomitant pruritus. Materials and methods. Patients were examined with vulgar PS (n = 97), which were divided into 2 groups: group 1 - PS with pruritus (n = 73, mean age 40.0 ± 1.5 years), group 2 (comparison group) - PS without pruritus (n = 24, mean age 41.5 ± 2.7 years). The cellular immunity indicators was carried out by flow cytofluorimetry. Phagocytic activity of peripheral blood neutrophils was studied microscopically by the absorption of latex particles. Concentrations of immunoglobulins, cytokines, circulating immune complexes in the blood serum were evaluated by the method of enzyme-linked immunosorbent assay. Statistica 6.0 was used for statistical analysis. Our studies allowed us to identify features of PS with concomitant pruritus: severe clinical course of the disease with damage to the musculoskeletal system, associated with selective deficiency of class M immunoglobulins and increased activity of phagocytic neutrophils.

Change in the Content of Immunoproteasomes and Macrophages in Rat Liver At the Induction of DonorSpecific Tolerance

Induction of donor specific tolerance (DST) by the introduction of donor cells into a recipients portal vein is one of the approaches used to solve the problem of transplant engraftment. However, the mechanism of DST development remains unclear to this moment. In the present work, we first studied the change in the content of immunoproteasomes and macrophages of the liver at early stages of the development of allospecific portal tolerance in rats by Western blotting and flow cytofluorimetry. On the basis of the data obtained, we can conclude that the induction of DST is an active process characterized by two phases during which the level of the proteasome immune subunits LMP2 and LMP7 in liver mononuclear cells, including Kupffer cells, and the number of Kupffer cells change. The first phase lasts up to 5 days after the beginning of DST induction; the second phase - from 5 to 14 days. In both phases, the level of the subunits LMP2 and LMP7 in the total pool of mononuclear cells and Kupffer cells increases, with maximum values on days 1 and 7. In addition, the total number of Kupffer cells increases in both phases with a shift in several days. The most noticeable changes take place in the second phase. The third day is characterized by a lower content of mononuclear cells expressing immunoproteasomes compared to the control value in native animals. Presumably, at this time point a window of opportunity appears for subsequent filling of an empty niche with cells of different subpopulations and, depending on this fact, the development of tolerance or rejection. The results obtained raise the new tasks of finding ways to influence the cellular composition in the liver and the expression of immunoproteasomes on the third day after the beginning of DST induction to block the development of rejection.

Culture cells in a model of microgravity

The main objective of the work was to clarify the question - how will cell cultures functional state change after microgravity simulation when the shift in full strength direction takes place? Proliferation processes and apoptosis intensity in cell lines of rat glioma and human fibroblasts were compared in changing the position of flasks with cell culture in relation to the horizon. The detection of apoptosis and necrosis processes was carried out using flow cytofluorimetry. It was found that the change in full strength direction provides an inhibitory effect on tumor glial cells and fibroblasts’ proliferative activity enhances along with inhibition of apoptotic processes. Intensification of apoptotic processes in glioma cells and attenuation of cell death processes in normal cells - fibroblasts - are the result of cell cooperation disturbance.

Local infiltration of lymphocytes in large bowel tissue cancer and chronic colitis.

e21101 Background: comparative analysis of concentrations of lymphocyte subpopulations in tissue of various intestinal zones in cancer of large bowel and chronic atrophic colitis. Methods: subpopulation composition of T- (CD3, CD4, CD8) and B- (CD19) lymphocytes, natural killers NK (CD16CD56), and T-lymphocytes with antigen-recognizing receptors TCRαβ and TCRγδ were examined in bowel adenocarcinoma tissue, peritumoural zone (1-3 cm away from tumour) and in resection line (10 cm away from tumour edge) and in chronic colitis tissue. Flow cytofluorimetry was used for immunophenotyping of the lymphocytes Results: 52 cases of adenocarcinoma and 24 cases of chronic colitis were examined. CD3 level in tumour tissue (64.2±3.6%) didn’t differ from this in colitis samples (65.6±3.5%); CD3 level was significantly lower in peritumoural zone and resection line (54.9±4.0% and 51.9±3.6%, correspondingly, p<0.05) than in colitis samples. The quantaty of lymphocytes with TCRαβ receptor in all tissue samples with cancer was significantly higher in comparison with one in chronic colitis samples. The level of lymphocytes with TCRαβ receptor in all tissue samples with cancer was 49.8±7.25, in peritumoural zone – 39.3±5.9%, in resection line 46.5±6.0%, compared to chronic colitis – 20.7±5.7%, p<0.05. Concentration of lymphocytes with TCRγδ receptor in cancer samples was 3-5 times less (tumour – 14.2±4.8%, peritumoural zone – 9.5±1.5%, resection line – 11.3+2.1%) than in samples colitis ones (42.9±9.9%) p<0.05. It was found that tumour tissue, in contrast to peritumoural tissue, accumulated T-lymphocytes (64.2±3.6% and 51.9±3.6%, correspondingly) due to CD4 cells (35.5±2.3% and 24.3±2.1%, correspondingly, p<0.05). Lower level of B-cells was found in tumour (18.0±2.8% and 30.1±3.7%, correspondingly, p<0.05). Conclusions: abnormalities of local cellular immunity at diseases of large bowel are probably related not only to imbalance of lymphocyte subpopulations, but also to change of local correlation of levels of αβ-T-lymphocytes capable of recirculation, and tissue γδ-T-lymphocytes. This may cause inadequacy of immune protection at cancer of large bowel.